Altering the immundominance hierarchy using a DNA vaccine expressing conserved regions

ABSTRACT

The invention provides methods and compositions for eliciting broad immune responses. The methods employ nucleic acid vaccines that encodes highly conserved elements from a virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 14/382,281, filed Aug. 29, 2014, which is a National Stage of International Application No. PCT/US2013/028932, filed Mar. 4, 2013, and which claims the benefit of U.S. Provisional Application No. 61/606,265, filed Mar. 2, 2012. Each of the forementioned applications is herein incorporated by reference for all purposes.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing as a text file named “077867-1011978-606200US-SEQLIST.txt” created Aug. 3, 2016 and containing 104,769 bytes. The material contained in this text file is incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

HIV-1 strains are highly variable and this diversity provides a major challenge for vaccine design. A candidate vaccine should provide protection against most clades of HIV. To address this problem, approaches to maximizing immunological strength and breadth are being explored, including strategies that use consensus, center-of-tree or ancestral sequences, multiple strains or mosaic immunogens, immunogens consisting of known epitopes from the database, and chimeric molecules expressing a selection of the most conserved epitopes from different clades of HIV [1-17, 56, 96].

In addition to sequence diversity, the presence of potential immunodominant epitopes provides another hurdle in the development of effective HIV vaccines. Accumulating evidence indicates that immunodominant epitopes exist, and that they may constitute an impediment for the production of effective universal HIV vaccines [18-31], as subdominant epitopes within HIV proteins have generally been associated with virologic control [19,22]. The use of any gene encompassing a complete protein as immunogen contains variable as well as conserved regions. Since variable sequences can mutate to escape immune responses while retaining function, and can contain immunodominant T cell epitopes, we argue that variable segments should be excluded from the design [32]. Our vaccine approach thus focuses on the induction of immune responses to nearly invariable proteome segments, many of which should be essential for the function of the virus, and the prevention of responses against variable segments and potentially immunodominant “decoy” epitopes [32-34].

The conserved element approach is supported by the following observations: (i) Viral proteins recover ancestral amino acid (AA) states when transmitted to a new host [35], and in the absence of the specific immune responses found in the previous host, they can recover a more fit state [36-38]; (ii) changes in conserved AA of viral proteins can destroy or significantly weaken HIV, indicating a critical role in virus biology [39-42]; (iii) CTL responses against specific viral proteins (e.g., Gag) are associated with relative control of viremia [43-50], and in the case of controllers and long-term non-progressors, high avidity CTLs targeting conserved regions have been identified [34, 51]; (iv) immunodominance of some epitopes can obscure or prevent reactivity against other, potentially protective epitopes [52]; (v) some AA segments in viral proteins are conserved throughout a given HIV-1 subtype, the entire group M, and, in some instances, in HIV-2 and SIV [32,53]. Together, these considerations predicted that an HIV vaccine that does not contain variable epitopes, and thus lacks potentially immunodominant decoy epitopes, but instead consists of strictly conserved proteome elements is better fit to induce immune responses able to prevent virus acquisition or virus propagation [32, 53]. The conserved elements used in our work differ from those used by others [11, 12, 16, 17, 54-56] that were selected using different criteria, as we have focused on both conservation and associations of particular sequences with immune control.

Previous work has been performed using Gag as a prototype vaccine, because Gag-specific T cell responses were found to correlate with control of viremia in clade B and C infected individuals [43, 48-50]. Seven highly conserved elements (CE) were identified in HIV-1 p24^(gag)[32, 34] (see also FIG. 1A). Indeed, a cross-sectional ex vivo study showed broad recognition of several CE in the context of wide HLA diversity and identified T cell responses of high functional avidity and broad variant reactivity [34], predominantly in controller individuals, suggesting an association between these T-cell responses and HIV control.

The present invention address the need for an improved protocol for inducing an immune response by providing a strategy based on employing DNA constructs encoding conserve elements in conjunction with constructs encoding the substantially full-length protein from which the conserved element vaccine is derived.

BRIEF SUMMARY OF THE INVENTION

The immunogenic regimens of the present invention focus on immune responses to proteome segments important to the function of a protein, e.g., a viral protein such as a lentiviral gag protein, and preclude responses against segments that absorb much of the host immune response, but which can mutate to escape immune responses while retaining function (often referred to in the art as “immunodominant decoys”) (32). A conserved element vaccine (CEvac) administered in accordance with the invention has properties of a universal vaccine against a virus, such as a lentivirus, e.g., HIV, and is able to induce immune responses to most or all circulating strains. In one embodiment, the invention provides a method of generating an immune response where the method comprises administering a p24^(gag) DNA vaccine that expresses conserved elements, e.g., from 3 to 7 conserved elements (CE), of HIV-1 p24^(gag) and excludes potential immunodominant or variable regions acting as potential decoy epitopes (32); followed by administration of a DNA vaccine encoding a full-length gag, e.g., p55^(gag).

In some embodiments, the invention provides a method of inducing broad immune response including those direct to the highly conserved elements, the method comprising vaccinating with CEvac DNA and gag DNA either sequentially or by co-immunization where responses are not produced upon vaccination with full-length p55gag immunogen. This vaccination approach overcomes the problem of diversity by generating cross-clade gag-specific immune responses and broadens the p55gag induced T cell and humoral immunity. In some embodiments, the vaccines are delivered as DNA vaccines, e.g., plasmid DNA vaccines. In some embodiments, the vaccines are delivered as adenovirus vaccines or vaccinia virus vaccine, or using another virus vector-based vaccination strategy.

In some embodiments, the invention provides vaccine compositions comprising a nucleic acid encoding six or seven highly conserved elements from p24gag. In some embodiments, the elements encoded by the nucleic acid are arranged collinearly. In some embodiments, the seven elements are separated by alanine linkers for efficient proteolytic cleavage. In some embodiments, DNA vectors are engineered to express the Core proteins (conserved element polypeptides comprising multiple conserved elements) only, to express secreted Core proteins having the N-terminal GM-CSF signal peptide (SPCore) or to express as a core fusion to the monocyte chemoattractant protein 3 (MCP3) chemokine to stabilize the protein expression and enhance secretion of the proteins, or to the lysosomal associated membrane protein 1 (LAMP-1) to direct the proteins to the lysosomal compartment including access to the MHC class II pathway.

In some embodiments, a method of the invention comprises administering a nucleic acid encoding a conserved element from a protein, e.g., a viral protein such as Gag, and a nucleic acid encoding a naturally occurring variant of the conserved element, where the variant differs from the conserved element by 1 amino acid, e.g., where the conserved element is 8 amino acids in length; or has at least 80%, typically at least 90% or greater sequence identity to the conserved element. In some embodiments, the variant may differ from the conserved element by 1, 2, or 3 amino acids. The nucleic acids encoding the conserved element and variant conserved element may be present on the same vector or encoded by different vectors. In some embodiments, the conserved element is from HIV gag. In some embodiments where a nucleic acid encoding a conserved element and a nucleic acid encoding at least one variant conserved element are administered, the conserved element and variant conserved element sequences together account for at least 80%, or at least 90%, typically at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of naturally occurring variants of the protein. In some embodiments, conserved elements immunogens are constructed based on “regional focus”, which will account for >80% of clade C HIV-1 or clade B HIV-1. Such an approach, for example, provides more specific vaccines.

The invention thus provides a methods of generating a broad immune response, the method comprising administering a nucleic acid encoding at least three conserved elements, typically at least 4, 5, or 6, or more conserved elements, from a protein of interest, to an individual and administering a nucleic acid encoding the full-length protein to the individual. In some embodiments the conserved element is from a highly diverse viral proteins, e.g., an HIV protein such as HIV gag or env.

In one aspect, the invention provides a method of inducing an immune response in a subject, the method comprising administering a first Gag conserved element nucleic acid that encodes a first Gag conserved element polypeptide (“CE1 polypeptide”) that comprises six conserved elements from Gag to the subject, wherein the conserved elements are from different regions of Gag, and further, wherein each conserved element is at least 12 amino acids in length, but less than 30 amino acids in length and the conserved elements are not contiguous; and administering a nucleic acid encoding a full-length Gag protein. In some embodiments, the conserved elements are from HIV-1 p24gag. In some embodiments, the first conserved element polypeptide comprises at least one conserved element that has an amino acid sequence set forth in SEQ ID NOS:1-7, 32, or 33. In some embodiments, the first conserved element polypeptide comprises at least two, three, four five, or six conserved elements that have an amino acid sequence set forth in SEQ ID NOS:1-7, 32, or 33. In one embodiment, the first conserved element polypeptide comprises conserved elements that each have a sequence set forth in SEQ ID NOS:1-7; or the first conserved element polypeptide comprises conserved elements that each have a sequence set forth in SEQ ID NOS:3-6, 32, and 33.

In some embodiments, the methods of the invention further comprise administering a second Gag conserved element nucleic acid that encodes a second Gag conserved element polypeptide (“CE2 polypeptide”) that comprises at least one variant of a conserved element contained in the first Gag conserved element polypeptide, wherein the variant in the 2nd polypeptide differs from the variant in the first polypeptide by 1, 2, or 3 amino acids. Typically, the variant conserved element differs from the conserved element by only 1 amino acid. In some embodiment, the conserved element and variant conserved element each have a sequence set forth in FIG. 1; or FIG. 13. In one embodiment, the first conserved element polypeptide comprises the sequence of SEQ ID NO:15 and the second Gag conserved element polypeptide comprises the sequence of SEQ ID NO:16. In some embodiments the first conserved element polypeptide comprises the sequence of p24CE1c and the second conserved element comprises the sequence of p24CE2c as shown in FIG. 13. In some embodiments the first conserved element polypeptide comprises the sequence of p24CE1d and the second conserved element comprises the sequence of p24CE2d as shown in FIG. 13. The first and second Gag conserved element nucleic acids can be administered sequentially or concurrently. In some embodiments, one or more of the conserved element polypeptides comprise a signal peptide, such as GM-CSF or MCP-3. In some embodiments, one or more of the conserved element polypeptides comprise a sequence that targets the protein for degradation, e.g., a LAMP sequence. In some embodiments, the first and second nucleic acid Gag conserved element polypeptides are encoded by the same vector. In some embodiments, the first and second nucleic acid Gag conserved element polypeptides are encoded by different vectors. The nucleic acids encoding the first and second conserved element polypeptides may be administered multiple times. In some embodiments, the nucleic acid encoding the full-length Gag protein is administered after a nucleic acid encoding a conserved element polypeptide, e.g., at least 2 weeks or 4 weeks, or longer after a nucleic acid encoding a conserved element polypeptide.

In a further aspect, the invention provides a method of inducing an immune response to an HIV gag protein, the method comprising

-   (a) administering: -   (i) a nucleic acid encoding a polypeptide comprising SEQ ID NO:15     and a nucleic acid encoding a polypeptide comprising SEQ ID NO:16 to     a subject; or -   (ii) a nucleic acid encoding a polypeptide comprising p24CE1c as     shown in FIG. 13 and a nucleic acid encoding a polypeptide     comprising p24CE2c as shown in FIG. 13 to the subject; or -   (iii) a nucleic acid encoding a polypeptide comprising p24CE1d as     shown in FIG. 13 and a nucleic acid encoding a polypeptide     comprising p24CE2d as shown in FIG. 13 to the subject; and -   (b) administering a nucleic acid encoding p55^(gag).     In some embodiments, the nucleic acid pairs set forth in (i), (ii),     or (iii) of step (a) are encoded by the same vector. In some     embodiments, the polypeptides are fused to a GM-CSF signal peptide.     In some embodiments, the nucleic acid encoding p55 gag is     administered at least two weeks after step (a).

In a further aspect, the invention provides a method of inducing an immune response to an HIV gag protein, the method comprising administering at least one nucleic acid encoding a conserved element polypeptide comprising a sequence set forth in SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SE ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:37, or SEQ ID NO:39 to the patient; and administering a nucleic acid encoding a full-length gag protein. In some embodiments, the full-length gag protein is administered at least 2 weeks after administering the nucleic acid encoding the conserved element polypeptide.

In a further aspect, the invention provides a method of inducing an immune response to a protein of interest, the method comprising administering a nucleic acid encoding a conserved element polypeptide, wherein the conserved elements are from the protein of interest and the polypeptide comprises at least three conserved elements, each of less than 30 amino acids in length where the conserved elements are joined by linkers; followed by administering a nucleic acid encoding the full-length protein, wherein the nucleic acid encoding the full-length protein is administered at least two weeks after the nucleic acid encoding the conserved element polypeptide.

In some embodiments the nucleic acid constructs encoding the conserved element polypeptides and full-length Gag polypeptide are administered intramuscularly by in vivo electroporation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, Panels A-D: Design of the p24CE DNA vaccine. (Panel A) Alignment of the HXB2 p24^(gag) protein sequence (SEQ ID NO:50) with the consensus clades A (SEQ ID NO:51), B (SEQ ID NO:52) and C (SEQ ID NO:53) and the Group M consensus (SEQ ID NO:54), the Group M Center-of-Tree (COT-M) (SEQ ID NO:55), and the 7 CE included in p24CE1 and p24CE2. The ‘toggle’ amino acid differences between the CE1 and CE2 sequences are indicated. (Panel B) Localization of CE within the hexameric p24^(gag) structure. The p24^(gag) structure is modified from Pornillos et al. [97] and shows the location of CE1-CE7 (red), the toggle AA (blue) and the AA not included in the CE (black). The crystal structure of the hexamer was obtained from the www http address ebi.ac.uk/pdbsum (Panel C) Kyte-Dolittle hydrophobicity plots for two different collinear arrangements of the CEs. (Panel D) The p24CE (p24CE1 and p24CE2) proteins are composed of 7 CE arranged collinearly and linked via amino acid linkers (AAAE=SEQ ID NO:42). The secreted SP-p24CE contains the GM-CSF signal peptide. MCP3-p24CE is a fusion protein with the Monocyte chemoattractant protein 3 (MCP-3) chemokine. LAMP-p24CE is a fusion with the lysosomal associated membrane protein 1 (LAMP-1). CE sequences shown for p24CE1: CE1, CE2, CE3, CE4, CE5, CE6, and CE7 (SEQ ID NOS:1, 2, 3, 4, 5, 6, and 7, respectively; CE sequences shown for p24CE2: CE1, CE2, CE3, CE4, CE5, CE6, and CE7 (SEQ ID NOS:8, 9, 10, 11, 12, 13, and 14, respectively)

FIG. 2: Expression of the p24CE plasmids upon transient transfection in cultured cells. Plasmid DNA (1 μg) expressing different variants of either p24CE1 (left panel) or p24CE2 (right panel) proteins were transfected in HEK293 cells. The cultures were harvested 24 hrs later and proteins from equal amounts ( 1/250) from the cell-associated (top panel) and extra-cellular (bottom panel) fractions were resolved on a 12% NuPAGE Bis-Tris gel and analyzed by Western immunoblot using a goat anti-p24^(gag) antiserum and visualized using enhanced ECL. The membrane containing the cell-associated fractions was also probed with anti-human pan actin antibody to control for equal loading of the samples.

FIG. 3, A-C: Cellular responses in p24CE DNA vaccinated C57BL/6 mice. Mice were vaccinated using in vivo EP with 20 μg of the indicated p24CE1 (left panel) or p24CE2 (right panel) DNA plasmids (A, B) or with SP-p24CE1 DNA (C). Splenocytes from individual animals were stimulated (A) with the Group M Consensus peptide pool (15-mer peptides overlapping by 11 AA), (B) with the COT-M peptide pool (10-mers overlapping by 9 AA) consisting of the matching peptides of p24CE1 and p24CE2 proteins, and (C) with peptide pools representing the clade A, B, and C p55^(gag) sequences (15-mer peptides overlapping by 11 AA), as described in Materials and Methods. The frequency of CE-specific IFN-γ producing CD4⁺ (open bars) and CD8⁺ (filled bars) T cells was determined by polychromatic flow cytometry. The mean and SEM are shown. Three experiments were performed and data from a representative experiment are shown.

FIG. 4: Mapping of the p24CE-induced cellular immune responses. Pooled splenocytes from C57BL/6 mice (N=5) vaccinated with the indicated p24CE1 (left panels) or p24CE2 (right panels) DNAs were stimulated with the Group M Consensus peptide pools (15-mers overlapping by 11 AA) spanning the individual CEs. The frequency of CE-specific IFN-γ producing T cells was measured. CD4⁺ (open bars) and CD8⁺ (filled bars) Gag-specific T cells are shown.

FIG. 5, A-D: Phenotypic and functional analysis of T cell responses generated by p55^(gag) and p24CE DNA vaccination. (A) Mice (N=5/group) were vaccinated 3 times (week 0, 3 and 6) with 20 μg of a plasmid expressing HXB2 p55^(gag) (clade B) or 20 μg of a mixture of plasmids expressing SP-p24CE1 and SP-p24CE2. The mice were sacrificed 2 weeks after the last immunization. Three independent experiments were performed and a representative experiment is shown. (B) Pooled splenocytes were stimulated with Clade A, B or C peptide pools (15-mers) spanning the p24^(gag) region (left panel) and the Group M consensus peptide pool (right panel). The frequency of the CD4⁺ (open bars) and CD8⁺ (filled bars) p24^(gag)-specific IFN-γ producing T cells was determined. (C) The splenocytes from the SP-24CE (left panel) and p55^(gag) (right panel) DNA vaccinated mice were stimulated with peptide pools specific for the individual CEs. The frequency of the CD4⁺ (open bars) and CD8⁺ (filled bars) CE-specific IFN-γ producing T cells was determined. (D) Plot overlays show the phenotypic and functional characterization of the antigen-specific T cells induced by SP-p24CE (left panels) and p55^(gag) (right panels) DNA vaccines upon stimulation with p24specific peptide pool. Total T cells recovered from the spleen are shown as grey contours, and the antigen-specific IFN-γ⁺ T cells are overlaid as red (CD4⁺ T cells) or black (CD8⁺ T cells) dots. The plots show the CD4/CD8 distribution (top panel), memory phenotype as determined by CD44/CD62L staining (middle panel) and TNFα/CD107a expression (bottom panel) among the T cells from vaccinated mice. The frequency of CD4⁺ (red) and CD8⁺ (black) IFN-γ⁺ T lymphocytes is shown.

FIG. 6, A-C: Humoral immune responses in p24CE DNA vaccinated mice. (A) Anti-HIV-1 p24^(gag) antibodies were measured in plasma from p24CE and p55^(gag) DNA vaccinated C57BL/6 mice by a standard clade B p24^(gag) ELISA. The graphs show absorbance (optical density, OD) and pooled plasma samples dilutions from mice vaccinated with the different p24CE1 plasmids (top panel), p24CE2 plasmids (middle panel), or p55^(gag) DNA (bottom panel). (B) Humoral responses induced upon SP-p24CE or p55^(gag) DNA vaccination in mice were analyzed by Western immunoblot assays. The membranes contain p24^(gag) protein collected from supernatants of HEK293 cells transfected with 5 μg of the infectious molecular clone pNL4-3 (lane 1) or the p24CE proteins collected from the cell-associated fractions of cells transfected with SP-p24CE1 and SP-p24CE2 plasmids (lanes 2 and 3, respectively). The membranes were probed with plasma (1:5000 dilution) from mice vaccinated with a mixture of SP-p24CE1&2 DNAs (top panel) or p55^(gag) DNA (bottom panel) followed by anti-mouse IgG-HRP labeled antibody and visualized by ECL. (C) Detection of humoral responses to full-length p55^(gag) in mice vaccinated with p24CE or p55^(gag) DNA by Western immunoblot assay. The p55^(gag) proteins were obtained from HEK293 cells transfected with 0.5 μg of RNA/codon optimized plasmids expressing unprocessed p55^(gag) from clades A, B and C or COT-M, respectively. The proteins were resolved on 10% NuPAGE Bis-Tris gels, and the membranes were probed with plasma (dilution 1:200) from mice immunized with DNAs expressing the secreted p24CE proteins SP-p24CE1 (top panel), SP-p24CE2 (middle panel) and p55^(gag) (bottom panel).

FIG. 7, A-D: p55^(gag) DNA vaccination of macaques induces poor CE-specific cellular immune responses. (A) Alignment of the amino acid (AA) sequence of the 7 CE represented in the p24CE1 and p24CE2 proteins with HXB2 p24^(gag) protein (SEQ ID NO:50). The toggled AA in each CE is shown. The numbering of the AA in HXB2 p24^(gag) protein is according to the HIV data base at the www site hiv.lanl.gov/. (B) Both p24specific and CE-specific T cell responses were measured at 2 weeks after the last vaccination from 11 macaques, which were immunized with plasmid DNA encoding HIV Gag. (C) Mapping of the individual CE-specific responses in 5 (of 11) macaques that had responses to CE (panel B). The frequency of IFN-γ⁺ T cells specific for each of the 7 CE is shown. Open bars: CD4⁺ T cells; filled bars: CD8⁺ T cells. (D). Dot plots showing the IFN-γ⁺ and granzyme B (GzmB) production of the CE-specific CD4⁺ (red) and CD8⁺ (black) T cells from the 5 macaques shown in panel C.

FIG. 8, A-C: CE-specific cellular immune responses upon vaccination of macaques with p24CE DNAs. (A) Macaques were vaccinated with p24CE DNA and the frequency of CE-specific T cells was measured 2 weeks after the 2^(nd) vaccination (EP2wk2). IFN-γ⁺ CD4⁺ (open bars) and CD8⁺ (filled bars) T cells are shown. (B) Mapping of individual CE-specific responses in the 6 immunized macaques from panel A. The frequency of IFN-γ⁺ CD4⁺ (open bars) and CD8⁺ (filled bars) T cells specific for each CE is shown. (C) Phenotypic characterization of the CE-specific CD4⁺ (red) and CD8⁺ (black) T cells with CD28⁺CD95⁺ (central memory) or CD28⁻CD95⁺ (effector memory phenotype) is shown in the top panel; IFN-γ⁺ and granzyme B expression is shown in the bottom panel.

FIGS. 9, A and B: Induction of broad and polyfunctional T cell responses in p24CE vaccinated macaques. (A) The number of CE recognized per animal in macaques vaccinated with p24CE DNA and p55^(gag) DNA are shown. (B) Frequency of CE-specific polyfunctional T cells was evaluated by their ability to produce IFN-γ, TNF-α, CD107a and granzyme B (GzmB). The data from one representative macaque from each group is shown: M437 (top panel) vaccinated with p24CE DNA; P574 (middle panel) vaccinated with p55^(gag) DNA. The pie charts (right) show the proportion of polyfunctional responses in these macaques. Frequency of IFN-γ⁺, TNF-α⁺, CD107a⁺ and GzmB⁺ polyfunctional CE-specific T cells (4 functions) as % of total T cells in macaques vaccinated with p24CE DNA and p55^(gag) DNA, respectively, are shown (bottom panel). Median values are indicated.

FIG. 10, A-C: Boosting of p24CE DNA primed macaques with p55^(gag) DNA increases CE-specific cellular responses. (A) Vaccination schedule of group 1 with p24CE plasmid DNAs (EP1, EP2) followed by the heterologous p55^(gag) DNA boost (EP3) and group 2 with p55^(gag) DNA (EP1, EP2) followed by the heterologous p24CE DNA boost (EP3). (B) Frequency of the CE-specific IFN-γ⁺ T cells in both groups before (EP2wk2) and after the heterologous boost (EP3wk2). (C) Frequency of IFN-γ⁺ TNF-α⁺CD107a⁺ GzmB⁺ polyfunctional CE-specific T cells (4-function) in groups 1 and 2 before and after the heterologous boost.

FIGS. 11, A and B: Mapping of CE-specific T cell responses before and after heterologous boost. The CE-specific responses were mapped as described for FIG. 2B. The plots show comparisons of the responses upon p24CE DNA vaccination followed by p55^(gag) DNA boost (group 1, A) and upon p55^(gag) DNA vaccination followed by p24CE boost (group 2, B). The percentage of IFN-γ⁺CD4⁺ (open bars) and CD8⁺ (filled bars) T cells specific for each CE is shown.

FIGS. 12, A and B: Humoral immune responses upon p24CE DNA vaccination are boosted by p55^(gag) DNA vaccination. (A) Reciprocal p24^(gag) binding antibody endpoint titers (log) measured in plasma by ELISA at the indicated time points for macaques in group 1 and group 2 before and after the heterologous boosts. (B) Western immunoblot analysis was used to test the reactivity of the vaccine-induced antibodies from macaques in group 1 and 2 to p24^(gag) and the p24CE proteins. The membranes contain either p24^(gag) protein (lanes 1 and 4), p24CE1 (lanes 2 and 5) or p24CE2 (lanes 3 and 6) and were probed with plasma from macaques from group 1 (dilution 1:2000) and group 2 (dilution 1:500) collected before (EP2wk2; lanes 1-3) and after (EP3wk2, lanes 4-6) the heterologous boost.

FIG. 13: Sequences and configurations of p24CEc polypeptides (SEQ ID NOS:46 and 47) and p24CEd polypeptides (SEQ ID NOS:48 and 49). p24CE1c contains CEs 8, 9, 3, 4, 5, and 6 (SEQ ID NOS:32, 33, 3, 4, 5, and 6, respectively); p24CE2c contains CEs 8, 9, 3, 4, 5, and 6 (SEQ ID NOS:40, 41, 10, 11, 12 and 13, respectively. p24CE1d contains CEs 9, 3, 4, 5, 6, and 8 (SEQ ID NOS:33, 3, 4, 5, 6, and 32, respectively). p24CE2d contains CEs 9, 3, 4, 5, 6, and 8 (SEQ ID NOS: 41, 10, 11, 12, 13, and 40, respectively.

FIG. 14a-14b : Plasmid map (14 a) and sequence (14 b) (SEQ ID NOS:17, 56, 18, 57, 58, 19, 56, and 58) of plasmid 306H that encodes p24 CE1+p24 CE2.

FIG. 15a-15b : Plasmid map (15 a) and sequence (15 b) (SEQ ID NOS:20, 59, 21, 60, 61, and 58) of plasmid 202H that encodes LAMP-p24CE2.

FIG. 16a-16b : Plasmid map (16 a) and sequence (16 b) (SEQ ID NOS:22, 61, 23, 57, 60, and 58) of plasmid 191H that encodes LAMP-p24CE1.

FIG. 17a-17b : Plasmid map (17 a) and sequence (17 b) (SEQ ID NOS:26, 62, 27, 59, and 58) of plasmid 230H that encodes MCP3-p24CE1.

FIG. 18a-18b : Plasmid map (18 a) and sequence (18 b) (SEQ ID NOS:28, 62, 29, 59, and 58) of plasmid 231H that encodes MCP3-p24CE2.

FIG. 19a-19b : Plasmid map (19 a) and sequence (19 b) (SEQ ID NOS:24, 56, 58, 25, and 58) of plasmid 235H that encodes SP-p24CE2.

FIG. 20: Illustrative regimens for administering conserved element Gag vaccines and full-length p55^(gag) vaccine.

FIG. 21: Illustrative data showing cellular immune responses before and after the boost. Cellular immune responses were measured with peptides (15-mer overlapping by 11 amino acids) spanning the complete p24^(gag).

FIG. 22: Analysis of the responses to individual CE. The responses to each CE were mapped in all the animals using CE-specific peptides (mixture of 10-mer peptide overlapping by 9 amino acids and 15-mer overlapping by 11 amino acids) for each CE. The number of CE that showed positive responses per animal are shown.

FIG. 23: Different vaccine strategies induced similar levels of p27^(gag) antibody responses. Binding antibody titers were measured in the plasma by ELISA.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A “conserved element” as used herein refers to a protein sequence that is conserved across a protein that has high sequence diversity in nature, e.g., a viral protein such as an gag. The conserved element need not have 100% sequence identity across the diversity of naturally occurring sequence of the protein, but the sequence variability in the naturally occurring sequences is low, e.g., less than 20%. In some embodiments, the sequence variability is less than 10%. A conserved element is usually eight amino acids, or greater, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. Typically a conserved element is less than 50 amino acids in length and often is less than 40 or less than 30 amino acids. In some embodiments, a conserved element is less than 25 amino acids in length.

A “nucleic acid vaccine” as used herein includes both naked DNA vaccines, e.g., plasmid vaccine, and viral vector-based nucleic acids vaccines that are comprised by a viral vector and/or delivered as viral particles.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081(1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term “nucleic acid” is used interchangeably with gene, cDNA, oligonucleotide, and polynucleotide. A “nucleic acid” encompasses RNA as well as DNA.

The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (a polypeptide sequence comprising conserved elements), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or can be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and oftentimes over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length or over the full-length of an amino acid or nucleic acid sequences.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST software is publicly available through the National Center for Biotechnology Information on the worldwide web at ncbi.nlm.nih.gov/. Both default parameters or other non-default parameters can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

The term “operably linked” refers to a functional linkage between a first nucleic acid sequence and a second nucleic acid sequence, such that the first and second nucleic acid sequences are transcribed into a single nucleic acid sequence. Operably linked nucleic acid sequences need not be physically adjacent to each other. The term “operably linked” also refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a transcribable nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the transcribable sequence.

Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” as used herein applies to amino acid sequences. One of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are conservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);     -   2) Aspartic acid (D), Glutamic acid (E);     -   3) Asparagine (N), Glutamine (Q);     -   4) Arginine (R), Lysine (K);     -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);     -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);     -   7) Serine (S), Threonine (T); and     -   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins         (1984)).

The terms “mammal” or “mammalian” refer to any animal within the taxonomic classification mammalia. A mammal can refer to a human or a non-human primate. A mammal can refer to a domestic animal, including for example, canine, feline, rodentia, including lagomorpha, murine, rattus, Cricetinae (hamsters), etc. A mammal can refer to an agricultural animal, including for example, bovine, ovine, porcine, equine, etc.

The terms “enhanced immune response” or “increased immune response” as used herein refers to an immune response to the conserved element nucleic acid and full-length, or substantially full length protein that are administered, where the immune response is increased in comparison to when only the conserved element vaccine or full-length protein is administered. An “enhanced immune response” may include increases in the level of immune cell activation and/or an increase in the duration of the response and/or immunological memory as well as an improvement in the kinetics of the immune response. The increase can be demonstrated by either a numerical increase, e.g., an increased in levels of antibody in a particular time frame, as assessed in an assay to measure the response assay or by prolonged longevity of the response.

The terms “treating” and “treatment” refer to delaying the onset of, retarding or reversing the progress of, or alleviating or preventing either the disease or condition to which the term applies, or one or more symptoms of such disease or condition.

An “antigen” refers to a molecule, typically a protein molecule in the current invention, containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response. The term is used interchangeably with the term “immunogen.” Normally, an epitope will comprise between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The term “antigen” includes both subunit antigens, (i.e., antigens which are separate and discrete from a whole organism with which the antigen is associated in nature), as well as inactivated organisms, such as viruses.

Introduction

The invention is based in part, on the discovery that administration of one or more nucleic acids encoding a polypeptide comprising conserved elements from a protein to a subject in conjunction with administration of a nucleic acid encoding the full-length protein, or substantially full-length protein, enhances the immune response to the conserved element sequences. The protein can be any protein, but is typically a viral protein that exhibits sequence diversity in naturally occurring variants. In some embodiments, the viral protein is a retrovirus protein, such as a lentiviral protein. In some embodiments, the viral protein is a retroviral Gag or Env protein.

In some embodiments, administration of the nucleic acid encoding the full-length protein, or substantially full-length protein, follows administration of a conserved element nucleic acid construct. Thus, the invention further provides methods of inducing an immune response comprising sequential administration of at least one conserved element nucleic acid construct followed by administration of a nucleic acid construct comprising substantially a full length protein from which the conserved elements are derived.

Conserved Element Nucleic Acid Constructs

Conserved elements of a protein sequence can be determined using known methods. For examples U.S. Patent Application Publication No. 20110269937, which is incorporated by reference, describes methods of evaluating protein sequences that exhibit natural variability to identify regions that are conserved using computational methods.

A conserved element nucleic acid construct is typically generated by linking nucleic acid sequences that encode multiple conserved elements that target conserved sequence that are present within all or a high percentage, e.g., at least 80%, at least 90%, or at least 95%, or greater, of the naturally occurring variants of the protein in a population. In typical embodiments, a conserved element is from a region of a protein that when mutated, has deleterious effects on the function of the protein. In typical embodiments, a conserved element does not comprise an amino acid sequence that does not occur in a naturally occurring variant, i.e., the conserved element does not contain amino acid substitutions that would result in a sequence that has not been identified in a naturally occurring variant.

In some embodiments a immunogenic compositions employed in the invention relates to a viral protein, e.g., a retrovirus protein such as Gag. Conserved elements of Gag have been identified (see, e.g., U.S. Patent Application Publication No. 20110269937; Rolland et al., PLoS Pathog 3: e157, 2007; Mothe et al., PLoS One 7: e29717, 2012).

In some embodiments, the nucleic acid construct encoding the conserved element polypeptide encodes a polypeptide that comprises at least one, two, three, four, five, six, or seven conserved elements set forth in FIG. 1. In some embodiments, the nucleic acid construct encodes a polypeptide that comprises at least 8, typically, at least 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, or more consecutive amino acids from the conserved elements set forth in FIG. 1; or in SEQ ID NOS:1-14, 32, 33, 40 and 41.

In typical embodiments, more than one nucleic acid construct encoding the conserved elements is used where one construct encodes a first set of conserved elements and the second construct encodes a second set of conserved elements where one or more elements, often each of the conserved elements, of the second set of conserved elements differs from the first set by 3 or fewer amino acids. The residues where the sequences differ, however, are at sites of naturally occurring variation, so that each of the conserved elements in the first and second sets corresponds to a naturally occurring protein sequence. In some embodiments, each element of the second set is at least 80% or at least 90% identical to the corresponding element in the first set of conserved sequences. The nucleic acid construct encoding the first set of conserved elements and the nucleic acid construct encoding the second set of conserved elements may be present in the same vector or different vectors.

Each conserved element useful for an immunogenic nucleic acid administered in accordance with the methods of the invention is typically fewer 30 amino acids in length. In some embodiments, the conserved element is less than 25, 24, 23, 22, 21, 20, or 15 amino acids in length. In some embodiments, the conserved element is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

In the present invention, the conserved elements contained within the conserved elements are not contiguous in the native protein sequence. The individual conserved elements are typically joined to one another in the nucleic acid construct by a peptide linker, such as an alanine linker. Linker sequences are well known in the art. Typical peptide linker sequences contain Gly, Ser, Ala and Thr residues. Useful linkers include glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers. In some embodiments, the linker is AA, AAAE (SEQ ID NO:42), AAAA (SEQ ID NO:43), AAK, AG, AA, LAK, AAK, AAAAL (SEQ ID NO:44), and the like.

The conserved elements may be present in any order in the construct, they need not occur in the order of the naturally occurring sequence. For example, a conserved element that occurs toward the N-terminus of a protein may be encoded at region of the construct encoding the C-terminal end.

In some embodiments, a nucleic acid encoding a conserved element polypeptide for use in the invention encodes a polypeptide that comprises the conserved elements set forth in SEQ ID NOS:1-7. In some embodiments, such a nucleic acid construct encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:15 (“Core1”). In some embodiments, a nucleic acid encoding a conserved element encodes a polypeptide comprising the conserved elements set forth in SEQ ID NOS:8-14. In some embodiments, such a nucleic acid construct encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO:16 (“Core2”). In some embodiments, a nucleic acid construct encoding a polypeptide comprising SEQ ID NO:15 is administered with a nucleic acid construct encoding a polypeptide comprising SEQ ID NO:16.

In some embodiments, a nucleic acid encoding a conserved element encodes a conserved elements set forth in SEQ ID NOS:3, 4, 5, 6, 32, 33, 40 or 41, or a variant thereof that differs by 1 amino acid. In some embodiments, a variant of SEQ ID NO:33 may differ at 1, 2, or 3 amino acids. In some embodiments, the conserved element polypeptide comprises a sequence set forth in FIG. 13.

In the present invention, a conserved element nucleic acid construct is administered in conjunction with the full-length protein, or substantially full-length protein, from which the conserved elements are obtained. In the context of the present invention, “substantially full-length” refers to the region of the protein that includes all of the conserved elements, i.e., a sufficient length of the naturally occurring protein is provided that includes all of the conserved elements that are used in the conserved element construct.

The nucleic acid encoding the full-length protein may be administered concurrently with the conserved element vaccine. In some embodiments, a full-length protein may be administered as the priming vaccine prior to administration of one or more conserved element constructs, which are administered as a boost. In preferred embodiments, one or more nucleic acids encoding conserved elements are administered as the prime and the nucleic acid encoding the full-length protein is administered as a boost. The boost is typically administered anywhere from two weeks to one, two, three, or four months, or longer, following administration of the initial vaccine.

Often, the nucleic acid constructs encoding the conserved elements and/or full-length protein are one or more purified nucleic acid molecules, for example, one or more plasmid-based vectors (“naked” DNA).

In some embodiments, the nucleic acid component may comprise vectors that encode the antigen of interest where the vector is contained within a virus. Viral delivery systems include adenovirus vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, poxviral vectors, or lentiviral vectors. Methods of constructing and using such vectors are well known in the art.

Recombinant viruses in the pox family of viruses can be used for delivering the nucleic acid molecules encoding the antigens of interest. These include vaccinia viruses and avian poxviruses, such as the fowlpox and canarypox viruses. Methods for producing recombinant pox viruses are known in the art and employ genetic recombination. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545. A detailed review of this technology is found in U.S. Pat. No. 5,863,542. Representative examples of recombinant pox viruses include ALVAC, TROVAC, and NYVAC.

A number of adenovirus vectors have also been described that can be used to deliver one or more of the nucleic acid components of the vaccine. (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). Additionally, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Retroviruses also provide a platform for gene delivery systems. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

Members of the Alphavirus genus, such as, but not limited to, vectors derived from the Sindbis, Semliki Forest, and Venezuelan Equine Encephalitis viruses, can also be used as viral vectors to deliver one or more nucleic acid components of the nucleic acid/protein combination vaccines of the invention. For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al., J. Virol. (1996) 70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S. Pat. No. 5,789,245, issued Aug. 4, 1998).

Expression Constructs Encoding Fusion Polypeptides Comprising a Degradation Signal or Signal Peptide Sequence

In some embodiments, a nucleic acids encoding a conserved element vaccine encodes a form in which the conserved element is fused to a sequence to enhance the immune response, such as a signal peptide sequence or a sequence that targets the protein for lysosomal degradation. Such embodiments typically results in enhanced immune responses in comparison to embodiments where the conserved element vaccine is not fused to a signal peptide or degradation signal.

Lysosomal Targeting Sequence

In other embodiments, signals that target proteins to the lysosome may also be employed. For example, the lysosome associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) include a region that targets proteins to the lysosome. Examples of lysosome targeting sequences are provided, e.g., in U.S. Pat. Nos. 5,633,234; 6,248,565; and 6,294,378.

Destabilizing sequences present in particular proteins are well known in the art. Exemplary destabilization sequences include c-myc aa 2-120; cyclin A aa 13-91; Cyclin B aa 13-91; IkBα aa 20-45; β-Catenin aa 19-44; β-Catenin aa 18-47, c-Jun aa1-67; and c-Mos aa1-35; and fragments and variants, of those segments that mediate destabilization. Such fragments can be identified using methodology well known in the art. For example, polypeptide half-life can be determined by a pulse-chase assay that detects the amount of polypeptide that is present over a time course using an antibody to the polypeptide, or to a tag linked to the polypeptide. Exemplary assays are described, e.g., in WO02/36806, which is incorporated by reference.

Variants of such sequences, e.g., that have at least 90% identity, usually at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to the sequences noted above, e.g., a LAMP degradation sequence, can be employed in this invention, e.g., for fusion to an HIV gag conserved element polypeptide.

Additional degradation signals that can be used to modify retroviral antigens, e.g., HIV antigens in accordance with the invention include the F-box degradation signal, such as the F-BOX signal 47aa (182-228) from protein beta-TrCP (Liu, et al., Biochem Biophys Res Comm. 313:1023-1029, 2004). Accordingly, in some embodiments, an expression vector for use in the invention may encode a fusion protein where an F-box degradation signal is attached to an HIV antigen, e.g., gag.

Targeting to the Proteasome and Other Degradation Signals

Many polypeptide sequences that target a protein for degradation are known in the art. One example of destabilizing sequences are so-called PEST sequences, which are abundant in the amino acids Pro, Asp, Glu, Ser, Thr (they need not be in a particular order), and can occur in internal positions in a protein sequence. A number of proteins reported to have PEST sequence elements are rapidly targeted to the 26S proteasome. A PEST sequence typically correlates with a) predicted surface exposed loops or turns and b) serine phosphorylation sites, e.g. the motif S/TP is the target site for cyclin dependent kinases.

Additional destabilization sequences relate to sequences present in the N-terminal region. In particular the rate of ubiquitination, which targets proteins for degradation by the 26S proteasome can be influenced by the identity of the N-terminal residue of the protein. Thus, destabilization sequences can also comprise such N-terminal residues, “N-end rule” targeting (see, e.g., Tobery et al., J. Exp. Med. 185:909-920.).

Other targeting signals include the destruction box sequence that is present, e.g., in cyclins. Such a destruction box has a motif of 9 amino acids, R1(A/T)2(A)3L4(G)5X6(I/V)7(G/T)8(N)9, in which the only invariable residues are R and L in positions 1 and 4, respectively. The residues shown in brackets occur in most destruction sequences. (see, e.g., Hershko & Ciechanover, Annu. Rev. Biochem. 67:425-79, 1998). In other instances, destabilization sequences lead to phosphorylation of a protein at a serine residue (e.g., Iκbα).

In some embodiments, a conserved element polypeptide of the invention is fused to a LAMP degradation sequence. For example, the methods of the invention may employ a polypeptide in which SEQ ID NO:15 or SEQ ID NO:16 is fused to a LAMP degradation sequence.

Expression Constructs that Encode Secreted Fusion Proteins

A secretory polypeptide in the context of this invention is a polypeptide signal sequence that results in secretion of the protein to which it is attached. In some embodiments, the secretory polypeptide is a chemokine, cytokine, or lymphokine, or a fragment of the chemokine, cytokine, or lymphokine that retains immunostimulatory activity. Examples of chemokines secretory polypeptides include MCP-3 and IP-10. In other embodiments, the secretory polypeptide is a polypeptide signal sequence from a secreted protein such as tissue plasminogen activator (tPA) protein, growth hormone, GM-CSF, a cytokine, or an immunoglobulin protein. Constructs encoding secretory fusion proteins are disclosed, e.g., in WO02/36806.

In some embodiments, the signal peptide is a GM-CSF sequence, e.g., a mammalian GM-CSF sequence such as a human GM-CSF signal peptide sequence.

In some embodiments, a secretory signal for use in the invention is MCP-3 amino acids 33-109, e.g., linked to IP-10 secretory peptide.

In some embodiments, a conserved element polypeptide is joined to a GM-CSF signal peptide sequence. For example, in some embodiments, the methods of the invention may employ a polypeptide comprising SEQ ID NO:15 fused to a GM-CSF signal peptide and/or a polypeptide comprising SEQ ID NO:16 fused to a GM-CSF signal peptide.

Similarly, an expression construct encoding a full-length polypeptide, e.g., a construct that encodes p55 gag, may also be modified with a degradation sequence and/or a secretory sequence. Moreover, more than one construct encoding the full-length polypeptide, e.g., p55gag may be administered. For example a construct in which p55 gag is fused to a signal polypeptide such as GM-CSF may be used in conjunction with a construct in which p55gag is fused to a degradation sequence, such as LAMP.

Additional Properties of Expression Constructs

Within each expression cassette, sequences encoding an antigen for use in the nucleic acid vaccines of the invention will be operably linked to expression regulating sequences. “Operably linked” sequences include both expression control sequences that are contiguous with the nucleic acid of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that promote RNA export (e.g., a constitutive transport element (CTE), a RNA transport element (RTE), or combinations thereof; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.

Any of the conventional vectors used for expression in eukaryotic cells may be used for directly introducing nucleic acids into tissue. Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors. Such regulatory elements include, e.g., human CMV, simian CMV, viral LTRs, and the like. Typical vectors may comprise, e.g., those with a human CMV promoter, bovine growth hormone polyA site and an antibiotic resistance gene for selective growth in bacteria.

Other expression vector components are well known in the art, including, but not limited to, the following: transcription enhancer elements, transcription termination signals, polyadenylation sequences, splice sites, sequences for optimization of initiation of translation, and translation termination sequences.

In some embodiments, the nucleic acid component may comprises one or more RNA molecules, such as viral RNA molecules or mRNA molecules that encode the antigen of interest.

In typical embodiments, the nucleic acid constructs are codon-optimized for expression.

In the present invention, a “nucleic acid” molecule can include cDNA and genomic DNA sequences, RNA, and synthetic nucleic acid sequences. Thus, “nucleic acid” also encompasses embodiments in which analogs of DNA and RNA are employed.

An immunogenic composition of the invention can be administered as one or more constructs. For example, where two sets of conserved elements are employed, e.g., conserved element polypeptides Core1 and Core2, or conserved element polypeptides p2CE1c and p24CE2c or conserved element polypeptides p2CE1d and p24CE2d, a nucleic acid construct can encode both sets, or each set may be encoded by a separate expression vector. Thus, the expression constructs administered in accordance with the invention may be administered as multiple expression vectors, or as one or more expression vectors encoding multiple expression units, e.g., a discistronic, or otherwise multicistronic, expression vectors. For example, an expression vector may be employed that encodes both SEQ ID NO:15 and SEQ ID NO:16 or multiple expression vectors may be employed where SEQ ID NO:15 is encoded by one vector and SEQ ID NO:16 is encoded by another vector.

Preparation of Immunogenic Compositions

In the methods of the invention, the nucleic acid component is often directly introduced into the cells of the individual receiving the immunogenic composition. This approach is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery technologies include, “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, and cationic lipid complexes or liposomes. The nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253 or pressure (see, e.g., U.S. Pat. No. 5,922,687). Using this technique, particles comprised solely of DNA are administered, or in an alternative embodiment, the DNA can be adhered to particles, such as gold particles, for administration.

In some embodiments, e.g., where a nucleic acid component of the invention is encoded by a viral vector, the nucleic acid component can be delivered by infecting the cells with the virus containing the vector. This can be performed using any delivery technology, e.g., as described in the previous paragraph.

In some embodiments, the immunogenic compositions of the invention are administered by injection or electroporation, or a combination of injection and electroporation.

Assessment of Immunogenic Response

To assess a patient's immune system during and after treatment and to further evaluate the treatment regimen, various parameters can be measured. Measurements to evaluate vaccine response include: antibody measurements in the plasma, serum, or other body fluids; and analysis of in vitro cell proliferation in response to a specific antigen, indicating the function of CD4+ cells. Such assays are well known in the art. For example, for measuring CD4+ T cells, many laboratories measure absolute CD4+ T-cell levels in whole blood by a multi-platform, three-stage process. The CD4+ T-cell number is the product of three laboratory techniques: the white blood cell (WBC) count; the percentage of WBCs that are lymphocytes (differential); and the percentage of lymphocytes that are CD4+ T-cells. The last stage in the process of measuring the percentage of CD4+ T-lymphocytes in the whole-blood sample is referred to as “immunophenotyping by flow cytometry. Systems for measuring CD4+ cells are commercially available. For example Becton Dickenson's FACSCount System automatically measure absolutes CD4+, CD8+, and CD3+ T lymphocytes.

Other measurements of immune response include assessing CD8+ responses. These techniques are well known. CD8+ T-cell responses can be measured, for example, by using tetramer staining of fresh or cultured PBMC (see, e.g., Altman, et al., Proc. Natl. Acad. Sci. USA 90:10330, 1993; Altman, et al., Science 274:94, 1996), or γ-interferon release assays such as ELISPOT assays (see, e.g., Lalvani, et al., J. Exp. Med. 186:859, 1997; Dunbar, et al., Curr. Biol. 8:413, 1998; Murali-Krishna, et al., Immunity 8:177, 1998), or by using functional cytotoxicity assays.

Viral Titer

Viremia is measured by assessing viral titer in a patient. There are a variety of methods of perform this. For example, plasma HIV RNA concentrations can be quantified by either target amplification methods (e.g., quantitative RT polymerase chain reaction [RT-PCR], Amplicor HIV Monitor assay, Roche Molecular Systems; or nucleic acid sequence-based amplification, [NASBA®], NucliSens™ HIV-1 QT assay, Organon Teknika) or signal amplification methods (e.g., branched DNA [bDNA], Quantiplex™ HIV RNA bDNA assay, Chiron Diagnostics). The bDNA signal amplification method amplifies the signal obtained from a captured HIV RNA target by using sequential oligonucleotide hybridization steps, whereas the RT-PCR and NASBA® assays use enzymatic methods to amplify the target HIV RNA into measurable amounts of nucleic acid product. Target HIV RNA sequences are quantitated by comparison with internal or external reference standards, depending upon the assay used.

Administration of DNA Constructs

The DNA vectors are formulated for pharmaceutical administration. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, including intranasal, intradermal, subcutaneous or intramuscular injection or electroporation, the carrier preferably comprises water, saline, and optionally an alcohol, a fat, a polymer, a wax, one or more stabilizing amino acids or a buffer. General formulation technologies are known to those of skill in the art (see, for example, Remington: The Science and Practice of Pharmacy (20th edition), Gennaro, ed., 2000, Lippincott Williams & Wilkins; Injectable Dispersed Systems: Formulation, Processing And Performance, Burgess, ed., 2005, CRC Press; and Pharmaceutical Formulation Development of Peptides and Proteins, Frkjr et al., eds., 2000, Taylor & Francis).

Naked DNA can be administered in solution (e.g., a phosphate-buffered saline solution) by injection, usually by an intra-arterial, intravenous, subcutaneous or intramuscular route. In general, the dose of a naked nucleic acid composition is from about 10 μg to 10 mg for a typical 70 kilogram patient. Subcutaneous or intramuscular doses for naked nucleic acid (typically DNA encoding a fusion protein) will range from 0.1 mg to 50 mg for a 70 kg patient in generally good health.

DNA immunogenic compositions can be administered once or multiple times. DNA vaccination is performed more than once, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more times as needed to induce the desired response (e.g., specific antigenic response or proliferation of immune cells). Multiple administrations can be administered, for example, bi-weekly, weekly, bi-monthly, monthly, or more or less often, as needed, for a time period sufficient to achieve the desired response.

The nucleic acid constructs in accordance with the invention are administered to a mammalian host. The mammalian host usually is a human or a primate. In some embodiments, the mammalian host can be a domestic animal, for example, canine, feline, lagomorpha, rodentia, rattus, hamster, murine. In other embodiment, the mammalian host is an agricultural animal, for example, bovine, ovine, porcine, equine, etc.

Immunogenic compositions containing the DNA expression constructs can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration.

In therapeutic applications, the vaccines are administered to a patient in an amount sufficient to elicit a therapeutic effect, e.g., a CD8⁺, CD4⁺, and/or antibody response to the HIV-1 antigens encoded by the vaccines that at least partially arrests or slows symptoms and/or complications of HIV infection. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the vaccine regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

Suitable quantities of DNA, e.g., plasmid or naked DNA can be about 1 μg to about 100 mg, preferably 0.1 to 10 mg, but lower levels such as 1-10 μg can be employed. For example, an HIV DNA vaccine, e.g., naked DNA or polynucleotide in an aqueous carrier, can be injected into tissue, e.g., intramuscularly or intradermally, in amounts of from 10 μl per site to about 1 ml per site. The concentration of polynucleotide in the formulation is usually from about 0.1 μg/ml to about 4 mg/ml.

The vaccine may be delivered in a physiologically compatible solution such as sterile PBS in a volume of, e.g., one ml. The vaccines may also be lyophilized prior to delivery. As well known to those in the art, the dose may be proportional to weight.

The compositions included in the regimen described herein for inducing an immune response can be administered alone, or can be co-administered or sequentially administered with other immunological, antigenic, vaccine, or therapeutic compositions.

Compositions that may also be administered with the vaccines include other agents to potentiate or broaden the immune response, e.g., IL-15, IL-12, IL-2 or CD40 ligand, which can be administered at specified intervals of time, or continuously administered.

The vaccines can additionally be complexed with other components such as peptides, polypeptides and carbohydrates for delivery. For example, expression vectors, i.e., nucleic acid vectors that are not contained within a viral particle, can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun.

Nucleic acid vaccines are administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Felgner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997), each of which is incorporated herein by reference. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

As noted above, immunogenic DNA compositions can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous routes. Administration of expression vectors of the invention to muscle and by electroporation can be a particularly effective method of administration, including intradermal and subcutaneous injections and transdermal administration. Transdermal administration, such as by iontophoresis, is also an effective method to deliver expression vectors of the invention to muscle. Epidermal administration of expression vectors of the invention can also be employed. Epidermal administration involves mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647).

The immunogenic compositions can also be formulated for administration via the nasal passages. Formulations suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, include aqueous or oily solutions of the active ingredient. For further discussions of nasal administration of AIDS-related vaccines, references are made to the following patents, U.S. Pat. Nos. 5,846,978, 5,663,169, 5,578,597, 5,502,060, 5,476,874, 5,413,999, 5,308,854, 5,192,668, and 5,187,074.

The vaccines can be incorporated, if desired, into liposomes, microspheres or other polymer matrices (see, e.g., Felgner et al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2nd ed. 1993). Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.

EXAMPLES ILLUSTRATING THE INVENTION Example 1. Core DNA Vaccination Induces Cross-Clade Specific Cellular Immune Responses in Mice

Results

Conserved Element DNA Vaccines

A set of conserved elements (CE) was identified in p24^(gag) composed of amino acids (AA) highly conserved across the entire HIV-1 group M, as determined by using the Los Alamos HIV database (www site is hiv.lanl.gov/) [32]. A refined list of 7 CE was selected based on several criteria (FIG. 1A, see Materials and Methods): a minimum length of 8 AA; inclusion of specific epitopes that have been correlated with viral control (low viral loads) in vivo; and exclusion of epitopes associated with high viral loads. The selected CE span 12-24 amino acids each, and together a total of 124 AA, thus representing 54% of p24^(gag) sequence. The CE are highlighted on the p24^(gag) capsid ribbon structure [97], revealing that they encompass most of the extended coiled regions of the p24^(gag) protein (FIG. 1B).

We generated multiple DNA-based vaccines in which the 7 CE were collinearly arranged (FIG. 1C), and connected via short linker sequences (FIG. 1D), designed for efficient proteolytic cleavage [91, 92]. Proteolytic processing of CE peptides in vitro revealed production of optimal epitopes or slightly extended optimal epitopes from 6 of the 7 CE segments (S. Le Gall, in preparation). Therefore, the CE immunogen is predicted to be able to present a significant number of native T cell epitopes after expression. For optimal arrangement of the different segments within the p24CE immunogens, the hydrophobicity of individual CE was taken into consideration (FIG. 1C). To avoid the strongly hydrophobic N-terminus in the arrangement CE1-2-3-4-5-6-7 (left panel), which could impact the intracellular trafficking of the protein, the CE1 peptide was placed at the C-terminus (right panel).

The majority of the AA included in the p24CE immunogens are essentially invariant since they are found in >98% of HIV isolates. The length of p24CE was expanded by including some less well-conserved AA (‘toggle’ sites), thus expressing additional potentially immunogenic regions. This allowed the extension of the 7 CE to the length of 12-24 AA, as mentioned above, and led to two p24CE sequences differing by 7 AA, one in each CE (FIG. 1A), named p24CE1 and p24CE2. These two sequences cover >99% of all known HIV-1 group M sequences. The p24CE1 and p24CE2 sequences were RNA/codon optimized [73-75] to maximize mRNA processing, transport, stability and translation (see Material and Methods). The p24CE coding regions were cloned into the pCMVkan vaccine vector (p24CE; FIG. 1D). Additional expression plasmids were generated in order to alter the intracellular trafficking and processing of the p24CE proteins. Plasmids SP-p24CE1 and SP-p24CE2 contain the GM-CSF signal peptide at the N-terminus of p24CE to promote secretion of the p24CE proteins. Plasmids MCP3-p24CE1 and MCP3-p24CE2 express fusion proteins with the monocyte chemoattractant protein 3 (MCP-3) chemokine, previously shown to stabilize the encoded protein and to enhance trafficking to antigen presenting cells [80, 81. Plasmids LAMP-p24CE1 and LAMP-p24CE2 express fusion proteins with the human lysosomal associated membrane protein 1 (LAMP-1). Fusion of Gag to LAMP was previously shown to direct it to the lysosomal compartment and to facilitate access to the MHC class II pathway as well as to the extracellular compartment [98-102].

Expression of the p24CE Proteins in Human Cells

The expression of the p24CE vectors shown in FIG. 1D was evaluated by Western immunoblots using cell extracts and supernatants from transiently transfected HEK293 cells (FIG. 2). To control for equal loading, the membrane containing the cell-associated samples were probed with an antibody against human beta actin as internal control (middle panel) demonstrating that similar amounts of proteins were loaded into each lane which validates our conclusions regarding the stability and different distribution of the proteins encoded by the different transfected plasmids (see below). Very low levels of the p24CE1 and p24CE2 proteins were detected in the cell-associated fractions (lanes 1 and 5, respectively), and no proteins were found in the extracellular compartment, indicating that the p24CE proteins were unstable. We also noted that p24CE2, differing only by 7 of the 124 AA from p24CE1, produced an even less stable antigen. The presence of the GM-CSF signal peptide (SP) greatly increased the levels of both p24CE proteins (lanes 2 and 6) in both the cell-associated and the extracellular fractions. These data indicate that the signal peptide altered the trafficking of the p24CE proteins, and promoted increase in stability and secretion. We noted the presence of additional bands of the secreted p24CE proteins, likely due to posttranslational modifications related to the altered cellular trafficking (compare lane 2 and lane 1; lane 6 to lane 5). The MCP3-p24CE (lanes 3 and 7) and LAMP-p24CE fusion proteins (lanes 4 and 8) were also readily detectable, and thus these fusions greatly stabilized the p24CE proteins. MCP3-p24CE localization in the extracellular fraction (lanes 3 and 7) as several bands, was similar to our previous report on a MCP3-Gag fusion protein [81]. The LAMP-p24CE proteins accumulated primarily in the cell-associated fraction (lanes 4 and 8), although some protein could also be found in the extracellular fraction, as we previously observed for the LAMP-p55^(gag) protein [101]. These data showed that altering the trafficking of the p24CE proteins, by adding the GM-CSF signal peptide or upon fusion to the MCP3 or LAMP molecules, enhanced the stability and modulated the trafficking of p24CE proteins.

Vaccination with p24CE Induces CE-Specific Cellular Immune Responses in C57BL/6 Mice

We next evaluated the immunogenicity of different p24CE proteins after DNA vaccination of C57BL/6 mice. Groups of mice (N=5) were vaccinated twice (0 and 4 weeks) with the indicated p24CE plasmids or sham plasmid DNA, as negative control, by intramuscular injection followed by in vivo electroporation (EP). Two weeks after the last vaccination (week 6), the mice were sacrificed and the presence of CE-specific cellular responses was determined by polychromatic flow cytometry. Splenocytes from the individual animals from each of the vaccine groups and the sham DNA inoculated negative control group were stimulated with a Group M consensus Gag peptide pool (15-mer peptides overlapping by 11 AA) (FIG. 3A) or with a COT-M peptide pool (10-mer overlapping by 9 AA) consisting of both p24CE1 and p24CE2 sequences (FIG. 3B). The use of the 15-mer peptide pool allowed for the detection of both CD4⁺ and CD8⁺ T cell responses, whereas the 10-mer peptide pool favors mainly CD8⁺ T cell responses. Vaccination with plasmids expressing p24CE or the secreted p24CE (SP-p24CE) proteins induced both CE-specific CD4⁺ and CD8⁺ T cell immune responses (FIG. 3A). In contrast, vaccination with the p24CE fusion proteins, MCP3-p24CE or LAMP-p24CE, elicited CE-specific responses that were almost exclusively mediated by CD4⁺ T cells. In agreement with these results, splenocyte stimulation with 10-mer peptide pools, which are mainly associated with MEW class I antigens, induced very low responses in MCP3-p24CE DNA vaccinated mice and no responses in the LAMP-p24CE immunized mice, which verified the previous conclusions (FIG. 3B). We hypothesize that altered intracellular trafficking of the p24CE fusion antigens could be responsible for the distinct preference for CD4⁺ or CD8⁺ T cell responses. Responses elicited by p24CE1 proteins were in general higher than those induced by p24CE2 (FIGS. 3A and 3B, note the different scales for p24CE1 and p24CE2), likely reflecting the higher expression of p24CE as indicated by the transient transfection experiments (see FIG. 2). As expected, no cellular responses were found in splenocytes from sham DNA vaccinated mice.

The cross-reactivity of the induced responses was analyzed using peptide pools representing different HIV-1 clades (A, B, and C; see also FIG. 1A). SP-p24CE1 DNA vaccination induced cross-clade reactive CD4⁺ and CD8⁺ cellular responses, which were similar in magnitude to those obtained with the Group M peptide pool (FIG. 3C). Cross-clade reactivity was also obtained upon vaccination with the other p24CE plasmids (data not shown). In contrast, splenocytes from mice immunized with sham DNA failed to recognize peptides from any of the three clade-specific peptide pools.

Fine Specificity of CE-Specific T Cell Responses from Vaccinated C57BL/6 Mice

Next, we assessed the distribution of the p24CE-induced cellular responses among the different CE (FIG. 4). Pooled splenocytes from the DNA vaccinated C57BL/6 mice (N=5/group) were stimulated with Group M consensus peptide pools (15-mer) encompassing the 7 individual CE. Polychromatic flow cytometry was used to determine the frequency of the CE-specific IFN-γ producing T cells and to discriminate between CD4⁺ and CD8⁺ T cell responses. Immunization with the different p24CE1 (left panels) and p24CE2 (right panels) plasmid DNAs induced cellular responses to CE1 and CE6, which were mediated almost exclusively by CD4⁺ T cells. Interestingly, mice immunized with plasmids encoding the native p24CE protein (p24CE and SP-p24CE) developed also high CD8⁺ mediated cellular responses to CE2. These data are in agreement with the cellular localization of the encoded proteins: the native p24CE protein remains mainly intracellular, while the SP-p24CE and MCP3-p24CE fusion are actively secreted and the LAMP-p24CE associates with the MEW class II compartment. Low levels of CD8⁺ T cell responses to CE3 were also identified upon immunization with the p24CE and the MCP3-p24CE plasmids. In conclusion, the p24CE proteins induced responses to 4 of the 7 CE (CE1, CE2, CE3, CE6) in mice, although these responses were generally lower in animals immunized with the p24CE2 plasmids, demonstrating that vaccination induced broad CD4⁺ and CD8⁺ T cell responses.

p24CE Induces Broader Immune Responses than the Full-Length p55^(gag)

We compared the immune responses to the individual CE upon vaccination with a p55^(gag) plasmid DNA or with a mixture of SP-p24CE1 and SP-p24CE2 DNAs. The mice (N=5/group) received 3 vaccinations (week 0, 3 and 6) and were sacrificed at week 8 (FIG. 5A). Vaccine-induced T cell responses were analyzed from pooled splenocytes (FIG. 5B) stimulated with 15-mer peptide pools specific for p24^(gag) of clade A, B, or C (left panel) and the group M consensus (right panel). The overall responses induced by the p55^(gag) immunogen were lower than those obtained by the p24CE immunogen, and remarkably, lacked CD8⁺-specific T cells. Using peptide pools spanning the individual CE (FIG. 5C) showed that p55^(gag) DNA elicited low responses to CE1 and CE6 only (right panel), mediated exclusively by CD4⁺ T cells. In contrast, vaccination with SP-p24CE DNA mixture elicited higher responses towards several CE (CE1, CE2 CE3 and CE6), as also expected from the data shown in FIG. 4.

We also evaluated the quality of the cellular immune responses elicited by the different immunogens (FIG. 5D) using the p24^(gag) peptide pool followed by intracellular cytokine staining and polychromatic flow cytometry. Vaccination with p55^(gag) DNA induced primarily CD4⁺ (red) T cell responses, while p24CE vaccination induced both CD4⁺ (red) (CE1 and CE6) and CD8⁺ (black) (CE2 and CE3) T cell responses (FIGS. 5B and 5C, top panel). Both immunogens induced effector memory T cells (CD44^(hi) and CD620^(neg)) (FIG. 5C, middle panel), which were mainly CD4⁺ (0.28% of total T cells) in mice vaccinated with p55^(gag) DNA, and both CD4⁺ (0.72% of total T cells) and CD8⁺ (0.37% of total T cells) in the mice vaccinated with the SP-p24CE DNA. Further analyses revealed that antigen-specific IFN-γ⁺ T cells produced TNF-α and expressed CD107a on the surface upon stimulation with antigen, indicating induction of cytotoxic T cells (FIG. 5D, bottom panel). We also noted that the CD8⁺ T cells (black), induced only by SP-p24CE DNA vaccination, expressed higher levels of CD107a and lower levels of TNFα than the CD4⁺ T cells (red), a phenotype consistent with the degranulation associated with CTL activity. Collectively our results show that the p24CE vaccine increased breadth and magnitude of cellular responses to p24^(gag) region in DNA vaccinated mice, by inducing robust responses to several of the highly conserved elements, and that the responses are multifunctional, a desired feature for an effective HIV vaccine.

Vaccination with p24CE Induces Cross-Clade Reactive Humoral Immune Responses

We next examined the induction of humoral immune responses using pooled plasma samples from p24CE DNA vaccinated mice (N=5/group) by an ELISA measuring clade B p24^(gag) responses (FIG. 6A). The different p24CE antigens readily induced high levels of humoral responses with titers similar or greater than those achieved in p55^(gag) DNA vaccinated mice (except p24CE2, middle panel). Vaccination with p24CE DNA induced antibodies to both the p24CE proteins (FIG. 6B, top panel, lanes 2 and 3) as well as to the processed p24^(gag) protein (lane 1). In contrast, the antibodies induced by p55^(gag) DNA vaccination readily detected p24^(gag) (FIG. 6B, bottom panel, lane 1), but failed to recognize the p24CE proteins (lanes 2 and 3). Thus, similar to the cellular immune responses (see FIG. 5), the antibodies elicited upon vaccination with full-length p55^(gag) DNA were unable to recognize the conserved elements.

We also examined the cross-clade reactivity of these responses by Western immunoblot analysis (FIG. 6C). Membranes containing p55^(gag) proteins from consensus clades A and C, clade B (HXB2) and COT-M, obtained from transiently transfected cells, were probed with pooled plasma samples from mice vaccinated with plasmids expressing SP-p24CE1, SP-p24CE2 or p55^(gag). The Western immunoblot assays showed that the antibodies induced by the p24CE and p55^(gag) DNA vaccinated mice detect the different p55^(gag) proteins. These data suggest that similar to p55^(gag), p24CE vaccinated mice induce cross-clade reactive antibodies.

Together, these data show that p24CE DNA vaccination induced strong humoral (FIG. 6) and cellular (FIG. 4) immune responses to the highly conserved elements in p24^(gag), and that CE segments are not or only poorly immunogenic when expressed as part of the complete p55^(gag) in DNA vaccinated C57BL/6 mice.

Discussion

The experiments performed in mice demonstrated that a DNA vaccine expressing 7 selected highly Conserved Elements within HIV-1 p24^(gag) can be produced and that this DNA vaccine is immunogenic in comparison to DNA-encoded full-length native p55^(gag). We have previously demonstrated that individuals chronically infected with HIV-1 develop cellular immune responses specific for the peptides encoded by the 7 conserved elements described in this work [34]. Furthermore, we found that the breadth, magnitude and avidity of these cellular responses to some CE were significantly higher among patients able to control HIV-1 infection, which suggests that responses against these conserved regions are clinically relevant [34].

Starting from our understanding of the rules for robust gene expression, and to avoid the escape potential of HIV, we constructed optimized DNA vectors that express maximal levels of new artificial immunogens based on highly conserved elements of the p24^(gag) region. This vaccine design is based on two principles, (i) the immunogen must include critical and highly conserved elements of the virus that cannot mutate without a severe loss in viability, and (ii) the immunogen must exclude HIV epitopes that are capable of mutating without significantly affecting viral fitness. The former may induce responses against a large number of HIV isolates, and the latter avoids immunodominant competition from variable regions, which may render ineffective the vaccine-induced immune response. Not only expression, but also the stability and presentation of the artificial antigens encoded by the DNA vectors were optimized. To this end, different fusion constructs were designed. We previously noted that either addition of a signal peptide or fusion to either MCP3 or LAMP were beneficial for protein expression [81, 80, 94], and found that these modifications also stabilize the p24CE proteins.

To maximize stimulation of CD4⁺ cells in addition to CD8⁺ T cells, we designed secreted immunogens. The p24CE antigen linked to the signal peptide of GM-CSF was expressed at high levels and also produced both CD4⁺ and CD8⁺ antigen-specific T cells as well as good antibody titers. In contrast, p24CE proteins fused to MCP-3 or LAMP directed the development of mostly CD4⁺ T cell responses. These studies show that it is possible to manipulate many properties of an antigen, altering the immune response in predictable ways. Although CD8⁺ T cell responses have been linked to control of viremia, we have also reported that cytotoxic CD4⁺ T cell responses contribute to viral control [103]. The p24CE vaccine induced both CD4⁺ (CE1 and CE6) as well as CD8⁺ (CE2 and CE3) specific T cell responses in the C57BL/6 mice; these CD8⁺ T cells had the functional phenotype of mature CTLs and were absent in mice immunized with the DNA encoding p55^(gag). The molecules generated allow the selection of the most optimal combinations to achieve the best protective response for HIV prophylaxis. We have found that p24CE proteins are more immunogenic than the full-length Gag protein, expanding the quality of cellular responses to recruit CD8⁺ T cells with the functional properties of canonical CTLs in C57BL/6 mice. These findings suggest that the peptides containing the CE regions produced from the full-length p55^(gag) antigen were not recognized efficiently by the T cells. This could be due to poor antigen processing or presentation, or alternatively due to interference by immunodominant peptides from other regions within p55^(gag), which are able to divert or inhibit immune response. In addition, as shown in FIGS. 3 and 4, the trafficking of the protein greatly affected its immunogenicity, with the p24CE and SP-p24CE eliciting the highest and most balanced CD4 and CD8 responses. Selection of the most optimal p24CE protein induced higher and broader immunogenicity than p55^(gag). We have recently also shown that dendritic cells in vitro loaded with RNA encoding the p24CE described in this work are able to stimulate T cell responses when mixed with autologous PBMC from HIV patients or to induce de novo T cell responses in PBMC from healthy donors. The responses elicited by p24CE were usually as high as those by full-length Gag [33].

All the immunogenicity studies described in the present work were performed in C57BL/6 mice. In our experience, p55^(gag) DNA immunization using the Balb/c mouse model induces higher T cell responses, but those responses are almost exclusively directed towards a single immunodominant epitope, AMQMLKETI (SEQ ID NO:45), which is present in our p24CE construct. Therefore, to avoid the restrictions imposed by this limited repertoire, we chose the C57BL/6 mouse model for the work described herein. We found very low primary immune responses to the CE regions after DNA vaccination using full-length p55^(gag). It will be of interest to further examine whether other vaccine modalities, i.e. recombinant viral vectors, expressing p55^(gag) are able to induce higher immune responses to the CE. To our knowledge, this study is the first comparative evaluation of immunity induced by a full-length immunogen and that induced by highly conserved elements from within the same protein. Our analysis points to the negative effect of regions outside of the defined conserved elements, which, it is important to note, are present in the full-length wild type as well as in the consensus and mosaic molecules as well as in the reported epitope immunogens. Thus, the use of the highly Conserved Element platform offers the advantage of focusing the immune responses to the invariable epitopes present in the viral proteome. Similar to the work described here, Letourneau et al. [11] previously demonstrated that a chimeric protein containing a string of several invariable regions from the HIV-1 proteome was immunogenic, but a direct comparison with the same sequences expressed within the natural proteins was not performed. In our study, we applied more stringent criteria to define conserved elements resulting in shorter peptide sequences (12-24 AA) that exclude adjacent more variable segments. In addition, our analysis of the immune responses was performed using peptide pools strictly confined to the conserved segments defined as immunogens and, therefore, the contribution to the T cell responses of putative artificial new epitopes created by the boundaries was completely excluded. In conclusion, we showed that the p24CE DNA vaccine induced broad cross-clade reactive cellular and humoral responses in vaccinated mice. We detected robust immune responses, including CD8⁺ T cells, to several CE upon p24CE DNA vaccination in mice, whereas only very poor (CD4⁺ only) or no responses to the CE were obtained by DNA vaccination with vectors expressing full-length p55^(gag). Thus, the inclusion of DNA vectors expressing the conserved elements is a promising vaccine strategy to induce broader immunity compared to vaccination with the p55^(gag) DNA alone. These results suggest further evaluation of the p24CE antigens in macaques.

Materials and Methods

p24^(gag) Conserved Elements Selection

Using all HIV-1 M group p24^(gag) coding sequences available in the 2009 Los Alamos database, we identified sequences of at least 8 AA in length, in which all AA were conserved in at least 98% of all sequences. This requirement was then relaxed in two ways: First, using available data that correlated epitope recognition with clinical viral load, we sought to include complete epitopes that were associated with low viral load and exclude epitopes that were associated with high viral load. Secondly, we allowed 1 toggle (variable) site/CE segment if the 2 most common AA at that site are together found in >99% of all known sequences [32]. To accommodate this variation, we created two plasmids, each with 7 CE segments from 12-24 AA in length, separated by 2-4 AA spacers (typically Ala-Ala-X) and differing only by the single toggle AA. The length and sequence of the spacers was set based on the existing knowledge of cleavage specificities and peptide availability [104], as well as to avoid fortuitous junctional homologies to HIV and the human proteome, the latter determined by searching against the HIV and human protein sequence databases.

DNA Plasmids

The p24CE and gag gene coding sequences were designed by RNA/codon optimization for efficient expression in mammalian cells [73-75] and chemically synthesized (GeneArt, Life Technologies, Grand Island, N.Y.). The genes were cloned into the pCMVkan vector [81] optimized for high gene expression. pCMVkan contains the human cytomegalovirus promoter, and the expressed transcripts contain a optimal surrounding for the AUG initiator codon from HIV-1 tat that prevents initiation of translation from internal AUGs [105], the bovine growth hormone (BGH) polyadenylation site, and the kanamycin resistance gene. This vector does not contain any splice sites or introns. The p24CE1 and p24CE2 proteins were produced from independent vectors (plasmids 164H and 182H, respectively). The secreted forms SP-p24CE1 and SP-p24CE2 contain the GM-CSF signal peptide (AA 1-17; Genbank accession Nr. NP 000749) at the N terminus (plasmids 234H and 235H). The MCP3-p24CE1 and MCP3-p24CE2 (plasmids 167H and 201H) are fusion proteins with the monocyte chemoattractant protein 3 (MCP-3) [80-81]. The LAMP-p24CE1 and LAMP-p24CE2 (plasmids 191H and 202H) are fusion proteins with the lysosomal associated membrane protein 1 (LAMP-1) [98-101]. Full-length p55^(gag) proteins were produced from RNA/codon optimized genes cloned into the pCMVkan plasmid, expressing Gag from clade A (plasmid 187H, Genbank accession number AAQ98129), clade B (plasmid 114H, HXB2, Genbank accession number AAB50258), clade C (plasmid 160H, Genbank accession number AAD12096) and the center-of-tree COT-M (222H) [106]. For immunizations HXB2 p55^(gag) was used. Endotoxin-free DNAs were prepared using Qiagen kit according to the manufacturer's protocol (Qiagen, Valencia, Calif.)

Transfection and Protein Analysis

DNA plasmids were transfected into 1×10⁶ HEK-293 cells using the calcium phosphate co-precipitation technique. Culture supernatants and cells were harvested 24 or 48 hours later, and protein expression was visualized by Western immunoblot analysis. The proteins were resolved on 10% or 12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, Calif.), transferred onto nitrocellulose membranes (Invitrogen), which were probed with a goat anti-p24^(gag) antibody (dilution 1:3000, provided by L. Arthur, SAIC, NCI, Frederick) followed by anti-goat IgG-HRP labeled antibody (dilution 1:10,000; Calbiochem, EMD chemicals, Gibbstown, N.J.) or with plasma (1:200 dilution) from DNA vaccinated mice followed by anti-mouse IgG-HRP labeled (1:10,000 dilution, GE Healthcare, Piscataway, N.J.). As control, the membranes were probed with anti-human pan-actin antibody (clone C4, EMD Millipore, Billerica, Mass.) at a dilution of 1:10,000. The bands were visualized using the enhanced chemiluminescence (ECL) plus Western blotting detection system (GE HealthCare, Piscataway, N.J.).

Mouse DNA Vaccination Studies

Female C57BL/6N (6 to 8 weeks old) were obtained from Charles River Laboratories, Inc. (Frederick, Md.) and were housed at the National Cancer Institute, Frederick, Md., in a temperature-controlled, light-cycled facility. The mice were immunized with 20 μg of the vaccine DNAs by intramuscular injection followed by in vivo electroporation by ELGEN® constant current electroporation device (Inovio Pharmaceuticals, Inc, Blue Bell, Pa.). As negative controls, a group of mice received equal amount of sham DNA following the same immunization protocol. The animals were vaccinated two (0 and 4 weeks) or three times (0, 3, and 6 weeks), and were sacrificed 2 weeks after the last vaccination when spleens and blood were collected for the analysis of cellular and humoral responses.

Intracellular Cytokine Staining

The frequency of antigen specific cytokine⁺ T cells was measured using polychromatic flow cytometry, as previously described [80]. The following set of 15-mer Gag peptide pools, overlapping by 11 AA, were used to stimulate the vaccine-induced cellular responses: HIV-1 consensus clade A (Cat #8116), consensus clade C (Cat #8118), and Group M Consensus (Cat #11057), obtained from the AIDS Research and Reference Reagent Program (Germantown, Md.); Gag 15-mer from HXB2/Clade B (Infinity Biotech Research & Resource, Inc, Aston, Pa.). Peptide pools spanning p55^(gag), p24^(gag) or only CE were generated. In addition, we used pools of 10-mer peptides overlapping by 9 AA from COT-M spanning the individual CE1-CE7, not including linker sequences, of p24CE1 and p24CE2 (peptide synthesis facility of the Massachusetts General Hospital, Boston). Splenocytes were cultured at 37° C. and 5% CO₂ at a density of 2×10⁶ cells/ml in complete RPMI-1640 medium containing Gag peptide pools at a final concentration of 1 μg/ml of each peptide. In all experiments, splenocytes cultured in medium without peptide pools or stimulated with phorbol myristate acetate (PMA) and calcium ionophore (Sigma, St. Louis, Mo.) were used as negative and positive control respectively. Protein secretion was blocked by the addition of monensin (GolgiStop, BD Biosciences) 1 hour after stimulation. After 12 hours incubation, the cells were harvested and cell surface staining was performed using the following antibody cocktail: CD3-APCCy7, CD4-PerCP, and CD8-Pacific Blue (BD Pharmingen, San Diego, Calif.). Splenocytes were washed twice, fixed, permeabilized with Cytofix/Cytoperm (BD Pharmingen) and staining for intracellular cytokine detection was performed using IFN-γ-FITC (BD Pharmingen). In another set of experiments, the antibody cocktail for surface staining included: CD3-AF700, CD4-PerCP, CD8-Pacific Blue, CD44-V500, CD62L-PE, CD107a-PE Cy7 (BD Pharmingen). The anti-CD107a antibody was added during the culturing of splenocytes with peptides. IFN-γ-APC and TNF-α-APC Cy7 (BD Pharmingen) were used for intracellular cytokine staining. After intracellular staining, the cells were washed twice and the samples were analyzed on an LSR II flow cytometer (BD Pharmingen). Data analysis was performed using the FlowJo platform (Tree Star, Inc., Ashland, Oreg.). All antigen specific responses are reported after subtracting values obtained from the samples without peptide stimulation. Only splenocytes giving a response more than two fold higher than the value of the sample without peptides (medium alone) were considered positive.

Antibody Assays

Serial dilutions of plasma samples were analyzed by standard HIV-1 clade B p24^(gag) ELISA (Advanced Bioscience Lab, Rockville, Md.), measuring optical absorbance at 450 nm.

Example 2. Core DNA Vaccination Induces Cross-Clade Specific Cellular Immune Responses in Macaques

Results

Vaccination with Gag DNA Induces Poor p24^(gag) Conserved Element (CE)-Specific Cellular Immune Responses in Macaques

We first investigated whether vaccination of macaques with a plasmid expressing p55^(gag) was able to elicit immune responses to the 7 CE [32, 34] identified within the p24^(gag) sequence (FIG. 7). Four animals were vaccinated twice (0, 2 month) with COT-M p55^(gag) DNA by IM injection followed by in vivo electroporation (EP). Seven animals previously vaccinated with a plasmid expressing the full-length p55^(gag) delivered intramuscularly (N=4) [82] or with a plasmid expressing the p37^(gag) protein delivered via IM/EP (N=3) [67] were included in the analysis. Induction of Gag-specific responses was evaluated upon stimulation of PBMC with a p24^(gag) specific peptide pool as well as with a CE-specific peptide pool (see Material and Methods). All 11 macaques developed readily detectable responses to the p24^(gag) region, however only 5 of the 11 macaques (45%) developed responses to CE (FIG. 7B). Next, we analyzed the specificity of the responses towards the individual conserved elements in the 5 macaques that showed CE recognition (FIG. 7C). We found that 3 macaques (L985, P574, R288) recognized only 1 CE (CE3 or CES), whereas 2 macaques (R067 and M121) developed responses to 2 CE (CE4, CES and CES, CE6, respectively). Moreover, only animal L985 developed significant CD8⁺ T cell responses against the CE, while the other 4 animals showed almost exclusively CD4⁺ T cell mediated responses. These CE-specific T cell responses included CD4⁺ and CD8⁺ T cells with cytotoxic potential, as judged by the presence of antigen-specific Granzyme B⁺ T cells in all 5 animals (FIG. 7D).

The lack of CE recognition in most of the vaccinated animals raised the concern that the immunogenicity of the CE epitopes within the Gag protein may be impaired due to either suboptimal processing and presentation of the CE-containing peptides, or immunodominance exerted by variable regions within Gag directing the CTL responses away from CE.

Vaccination with p24CE DNA Induces Cellular Immune Responses in Macaques

To test whether broader immune responses to the CE could be elicited in macaques, we vaccinated animals with a mixture of the two p24CE DNA vectors which were engineered to express the 7 CE collinearly arranged. (See Example 1). The two proteins differed by 1 AA (toggle') per CE, (SP-p24CE1 and SP-p24CE2) (FIG. 7A). Four animals were vaccinated twice (0, 2 month) with p24CE DNA using IM/EP delivery. Cellular immune responses were measured in blood samples collected 2 weeks after the 2′ vaccination (EP2wk2). Two additional macaques (M437, P314), previously immunized with p24CE plasmids, were also included in this analysis. All 6 macaques developed CE-specific cellular responses (FIG. 8A), as measured by the production of IFN-γ with a frequency ranging from 0.1% to 0.6% of total T cells. The overall levels of responses were non-significantly lower compared to those of the p55^(gag) DNA vaccinated animals (data not shown). These responses included both CD4⁺ and CD8⁺ T cells, although the CD8⁺ T cell responses were dominant in 4 of the 6 vaccinated animals (FIG. 8A). These results are in contrast to those obtained upon p55^(gag) DNA vaccination, where only 5 out 11 immunized animals developed CE responses, mediated mainly by CD4⁺ T cells (FIG. 7). Mapping of the CE-specific responses (FIG. 8B) revealed recognition of all CE except CE1 and CE7, using out-bred animals with different MEW class I haplotypes. Comparison of animals vaccinated with p24CE or Gag DNA shows that there was no apparent correlation between haplotype and ability to develop responses to CE, hence the differences could be attributed to the immunogen. Five of the 6 CE vaccinated macaques developed responses to 3 CE and only one animal (M437) showed responses to 1 CE. Phenotypic analysis of the antigen-specific T cells revealed both central (CD28⁺CD95⁺) and effector memory (CD28⁻CD95⁺) (FIG. 8C, top panels). A subset of the CE-specific IFN-γ⁺ T cells also expressed granzyme B, indicating a cytotoxic phenotype (FIG. 8C, bottom panels), thus eliciting cytotoxic CE-specific responses. These results indicate that, similar to our observation from vaccinated mice (Example 1), the CE DNA vectors are immunogenic in all 6 macaques and that most (5 of 7) of the CE were immunogenic. These data also demonstrate that CE-containing peptides are processed and presented properly and suggest that the failure to induce CE-specific responses from p55^(gag) or p37^(gag) is likely the result of immunodominance exerted by epitopes located in the variable regions.

Vaccination with p24CE DNA Induces Broader and Higher Levels of Polyfunctional CE-Specific T Cell Response than Vaccination with p55^(gag) DNA

We further dissected CE immunogenicity by comparing the cellular responses induced by p24CE and p55^(gag) DNA vaccination. First, we compared the number of CE recognized in the macaques vaccinated with DNA expressing the p24CE (N=6) or full-length p55^(gag) or p37^(gag) (N=11) (FIG. 9A). Immunization with p24CE induced responses to significantly more CE (p=0.0006; range 1-3 CE, median 3) than Gag DNA vaccination (range 0-2 CE) (FIG. 9A). These data demonstrate that p24CE DNA induced responses to more CE, indicating increased breadth of responses compared to p55^(gag) DNA vaccination.

We also compared polyfunctionality (production of IFN-γ, TNF-α, CD107a and granzyme B) of the T cell responses upon stimulation with CE-specific peptides. FIG. 9B shows the distribution of CE-specific polyfunctional T cells from representative macaques that received either p24CE DNA (top panel) or p55^(gag) DNA (middle panel). The proportion of polyfunctional T cells (1- to 4-function) is also shown as pie charts (right panels). These results demonstrate that p24CE DNA vaccination elicited higher CE-specific cytotoxic T cell levels than p55^(gag) DNA vaccination. The frequency of CE-specific T cells secreting two cytokines, expressing granzyme B and able to degranulate upon antigen recognition (4-function) was also significantly higher (p=0.03) in macaques immunized with p24CE DNA (bottom panel). Together, these data show that the p24CE immunogen elicited significantly higher responses, including to more CE, and that these responses are multifunctional and have cytotoxic properties.

p55^(gag) DNA Vaccination Boosts Pre-Existing CE-Specific T Cell Responses

Given that repeated vaccination with p55^(gag) DNA failed or only poorly induced de novo CE-specific T cell responses (FIG. 7), we investigated whether full-length gag DNA vaccination could boost and/or broaden pre-existing CE-specific immunity. The p24CE-vaccinated macaques received an additional vaccination with a plasmid expressing COT-M p55^(gag) DNA (FIG. 10A; group 1). This led to a significant increase (p=0.002) in CE-specific responses, reaching in some animals more than 1-2% of the total T cell population (FIG. 10B). Analysis of the polyfunctionality of these responses showed that the frequency of CE-specific T cells with 4 functions was also significantly boosted (p=0.002; FIG. 10C). Boosting with p55^(gag) DNA also induced de novo responses to p17^(gag) and C-terminal regions of Gag, thereby increasing the total Gag responses to levels similar to those obtained with the gag/p24CE DNA vaccine. Additionally, virtually the complete set of pre-existing responses to individual CE was boosted in all 6 macaques (FIG. 11A, left panel). The number of the CE found to be immunogenic upon p24CE vaccination (1-3 CE/animal) increased to 2-4 CE/animal upon Gag DNA boost. These findings confirmed that the suboptimal responses induced by priming with full-length Gag were not related to the absence of processing or presentation of CE-containing peptides, but rather to their inability to induce de novo responses in the presence of other, likely more dominant, Gag epitopes outside of CE. Thus, the immunodominance exerted by Gag epitopes outside of CE was lost in the presence of pre-existing CE-specific responses.

We also investigated whether p24CE DNA vaccination could alter the CE-specific immunity in macaques previously vaccinated with p55^(gag) DNA (FIG. 10A; group 2). Vaccination with p24CE DNA minimally increased the pre-existing CE-specific responses in 3 out of 4 macaques, (FIG. 10B, group 2) and modestly increased the polyfunctional CE responses in two of the vaccinated animals (FIG. 10C, group 2), although this increase was not statistically significant. Analysis of the individual CE (FIG. 11B) showed no new CE responses upon p24CE boost. The heterologous p24CE DNA boost did not alter the pre-existing CD4⁺ or CD8⁺ T cell distribution. Thus, the immunodominance exerted by epitopes outside of CE could not be overcome by p24CE vaccination, as this vaccine regimen did not alter the magnitude or breadth of Gag responses.

p24CE DNA Vaccination Induces Humoral Immune Responses that Recognize p24^(Gag)

The development of Gag-specific humoral immune responses was also monitored over the course of study (FIG. 12A) using a p24^(gag) ELISA. Upon vaccination with p24CE DNAs (group 1) antibodies recognizing p24^(gag) were readily detectable and peaked 2 weeks after EP2 (mean reciprocal end-point dilution titer 5.2 log). Similarly, the p24^(gag) antibody titers upon COT-M p55^(gag) DNA vaccination also peaked 2 weeks post EP2 (mean reciprocal end-point dilution titer 5.3 log). Thus, both vaccines elicited similar p24^(gag) antibody titers.

We further assessed the ability of these antibodies to recognize the p24CE proteins as well as processed p24^(gag) by Western immunoblots. The data from two representative macaques from each group are shown (group 1: L862 and M166, and group 2: P574 and R288) with similar data obtained from all animals from both vaccine groups. Plasma from macaques vaccinated with p24CE (Group 1) recognized naturally processed p24^(gag) produced from a clade B molecular clone of HIV-1 (FIG. 12B, lane 1) as well as the p24CE1 (lane 2) and p24CE2 (lane 3) proteins. In contrast, vaccination with p55^(gag) DNA (group 2) induced antibodies that strongly react with p24^(gag) (lane 1), but failed to recognize p24CE proteins (FIG. 12B lanes 2 and 3). We conclude that only the p24CE DNA vaccination induces robust cellular and humoral responses to the conserved elements.

Lastly, we tested whether boosting with the heterologous DNA (EP3) affected the pre-existing humoral immune responses. ELISA assays showed similar increase in p24^(gag) antibody levels in both groups (FIG. 12A). All Western immunoblot assays (FIG. 12B) were performed in parallel using the same plasma sample dilution and the same exposure time of the membrane to allow comparison of before and after the respective boosts. Following p55^(gag) DNA boost of the p24CE DNA vaccinated animals (group 1), stronger reactivities to both p24^(gag) (lane 4) as well as p24CE proteins (lanes 5 and 6) were found. These data demonstrate that p55^(gag) DNA vaccination was able to substantially boost the CE-primed humoral immune responses despite its failure to induce de novo antibody responses able to recognize the CE protein. Vaccination of the p55^(gag) DNA primed animals with p24CE DNA (group 2, bottom panels) showed induction of antibodies to p24CE proteins (lanes 5 and 6) and increased reactivity to p24^(gag) (lane 4). Note, a significantly higher amount of plasma was used in order to detect p24CE proteins from the animals in group 2 (dilution 1:500) compared to group 1 (dilution 1:2000). Thus, these data indicate that the heterologous p24CE DNA boost induced low level CE-specific responses, rather than inducing anamnestic responses (group 2). Together, these data show that prime immunization with p24CE DNA can alter the immunodominance of both cellular and humoral immune responses and that the immunodominance of epitopes outside of CE is not overcome by boosting with CE if the animal's vaccination involved priming with p55^(gag). Therefore, priming with p24CE DNA followed by the heterologous p55^(gag) DNA boost is a preferred approach to achieve broad and high cellular and humoral immune responses to the highly conserved elements of HIV-1 p24^(gag) protein.

Discussion

We have described DNA vectors encoding collinearly 7 highly conserved elements of the HIV-1 Group M p24^(gag) protein, and we have reported that vaccination of mice with these DNAs induced both cellular and humoral responses [57]. In the current report, we demonstrated that vaccination of rhesus macaques with these DNA vectors induced CE-specific cellular and humoral immune responses. Detailed analysis of cellular immune responses showed that p24CE DNA vaccination induced cytotoxic CD4⁺ and CD8⁺ T cells against CE and that the elicited T cell responses were polyfunctional. Therefore, our conserved element DNA vectors show desired features for an effective vaccine. Our vaccine regimen also shows a promising approach to overcoming a problem in the HIV vaccine field, where attempts to induce both antigen-specific CD4⁺ and CD8⁺ T cell responses and to broaden the vaccine-induced immunity to include subdominant epitopes have been less successful, even with a reported EP DNA/Ad boost immunization strategy [83].

Importantly, we found that the p55^(gag) vaccine elicits no or only poor responses to CE. We also analyzed the responses from a previous report [4], where macaques were vaccinated with consensus or mosaic p55^(gag) DNA as prime followed by recombinant Adenovirus boost. We found that 5 of the 12 animals that received the consensus molecule and 6 of 12 that received the mosaic molecules developed CE responses ranging from of 0-2 (consensus) and of 0-4 (mosaic) CE responses/animal, whereas several epitopes outside the CE were immunogenic in all the animals. In the study reported herein, we found that 5 of 11 macaques vaccinated with full-length COT-M or HXB2 p55^(gag) DNA developed CE-specific responses (0-2 CE/animal), whereas epitopes outside the CE were immunogenic in all the macaques. The data of the two studies are thus in good agreement, although the methods of analysis were not identical [peptide mapping [4] versus analysis with CE-specific mixture of 15-mer and 10-mer peptides (this report)]. Irrespective of the nature of Gag vaccine (consensus, mosaic or wild type), we found responses to CE in only 42-50% of the animals, and the responses were to very few CE/animal, suggesting that immunodominant epitopes within Gag focus the CTL response away from these conserved targets. In this report, we experimentally tested this hypothesis and demonstrated that immunodominance of variable regions is indeed responsible for the poor immunogenicity of the CE.

Although vaccination with either p55^(gag) or p37^(gag) induced strong humoral responses, we found that these antibodies fail to cross-react with the CE protein. In contrast, our engineered p24CE DNA vaccine readily induced both antibodies and cell-mediated responses to several CE, bypassing the restriction associated with full-length Gag vaccination. Importantly, immunizing with a full-length Gag greatly boosted the pre-existing CE responses. Hence, exposure to virus might also have the effect of boosting CE responses in CE-vaccinated individuals.

In a recent paper, Stephenson et al. [17] compared responses of full-length molecules to their conserved elements (Gag, Pol, and Env) vaccine and concluded that the conserved element vaccine did not provide any benefit (breadth or magnitude). In contrast, we demonstrated a clear benefit from the CE vaccine, showing increased breadth and magnitude of responses. The difference between the studies may substantively be due to our more strict definition and selection of CE, which, in contrast to others, were selected in part by their association with virus control [34], further supporting their immunological relevance.

Previous analyses of HIV-1 infected persons with different HLA haplotypes demonstrated the presence of CE-specific T cells during the chronic phase of infection [20, 34]. Higher avidity CTL responses in these regions were identified in HIV controllers and detailed analysis of the responses demonstrated that, for most epitopes analyzed, controllers were able to recognize more peptide variants [34]. This indicates that TCR promiscuity could be beneficial for the recognition of epitopes with mismatched amino acids resulting in better control of viral replication and prevention of escape mutants. These data also suggest that high avidity CE-specific responses are a potential correlate of HIV control. It is not clear why vaccination with full-length Gag generates poor CE responses (in mice or macaques), while these responses are detected in chronic HIV infection. It would be of interest to study different vaccination regimens and also to examine the time of development of CE responses during natural infection. The difference in elicited immune response is reminiscent of a previous report by Ferrari et al. [84], who showed that the immunodominant p17^(gag) SL9 response identified in HLA-A*0201 infected persons could not be induced upon ALVAC-gag vaccination in these haplotype-selected volunteers, although this epitope has been implicated in the Sieve effect observed in the STEP HIV vaccine trial [85]. Both studies suggest that there may be differences between vaccine-induced and infection-induced cellular responses that should be taken into consideration for successful vaccine design; they also highlight the potential immunodominant decoy effect of a full-length immunogen design.

Impaired immunogenicity of the conserved elements in the context of the natural protein sequence could be due to the presence of variable regions, which may exert an immunodominant decoy effect preventing the recognition of the conserved epitopes. This possibility is supported by a recent study where a bias was found towards less-conserved regions in HIV-1 Ad5 gag/pol/nef vaccinated human volunteers [86]. Generation of responses mainly outside of the conserved elements by full-length Gag suggested an immunodominant decoy effect. In this context, the success of our p24CE DNA prime-p55^(gag) DNA boost vaccine strategy is of great importance, because it showed strong boosting of pre-existing CE-specific cellular and humoral responses in macaques. Upon gag DNA boost, we report both a robust increase of the pre-existing CE-specific responses as well as development of de novo responses to regions outside the CE. These data imply that the immunodominance exerted by Gag epitopes outside of CE was lost in the context of pre-existing CE-specific responses.

The impaired immunogenicity of the conserved elements when expressed in the context of the complete Gag could in principle be related to suboptimal processing and presentation of the CE peptides, preventing efficient priming of adaptive immune responses. However, our p24CE prime-Gag boost study clearly demonstrates that processing of the full-length Gag protein produces a collection of CE-containing peptides that are recognized by T cells. We speculate that recognition of MHC-peptide complexes is less stringent for boosting memory T cell clones than for priming naïve T cells. Similar to the observations on cellular immunity, full-length Gag boosted pre-existing B cell responses, while failing to prime the development of de novo antibodies able to recognize the CE protein. These findings support the concept that proper processing of CE-containing peptides from the native Gag protein takes place, and that these sets of CE containing peptides are able to potently augment pre-existing responses to different extents. Together, these findings point to a critical difference in T cell recognition of these peptides where a clear distinction between antigen-experienced and naïve T cells is noted.

The question then arises whether a T cell vaccine can benefit from the responses elicited by selected T cell epitopes. A previous report [87] demonstrated the potency of T cell immunity in the absence of Env. In fact, a recent paper by Mudd et al. [88] showed that a T cell vaccine that induced Mamu-B*08-restricted CD8⁺ T-cell responses targeting 3 different viral epitopes elicited responses able to control SIV_(mac239) replication. Since our CE DNA vaccine was selected to highly restricted sequences and haplotype-independent, it is plausible that they too could induce such potent responses, which will be addressed in future studies.

The presented results contribute significantly to the development of improved vaccine candidates against HIV targeting the immune responses to essential highly conserved regions for the virus. We hypothesize that cellular immune responses targeting conserved regions of HIV and other highly variable pathogens, which do not allow rapid escape mutations without significant loss of viral fitness, are more likely to be protective [32-34]. Since there is evidence that vaccine-induced responses can change upon HIV infection resulting in virus escape in humans [89], a selection of strictly conserved elements is of great importance for the design of an effective vaccine. Such a selection should also avoid epitopes that may act as immunodominant decoys. Thus, a successful vaccine should be able to generate potent cross-clade specific humoral and cellular responses against conserved regions of the virus. Our results provide an effective strategy to overcome restrictions associated with immunodominance, while improving the magnitude and breadth of responses, especially those against conserved regions, minimizing the possibility of viral escape while increasing the recognition of naturally occurring divergent HIV strains. These results indicate that a vaccine candidate should be designed to extend this concept to the entire HIV proteome. Since the macaque model was in general shown to provide a similar response hierarchy to that obtained upon vaccination of humans comparing different vaccine platforms [90], our macaque study supports the evaluation of the novel CE vaccine strategies in humans.

Materials and Methods

DNA Vectors

The p24CE plasmids pSP-p24CE1 (plasmid 234H) and pSP-p24CE2 (plasmid 235H) have been described [57] and contain the human GM-CSF signal peptide at the N-terminus of the expression-optimized p24CE open reading frame. Briefly, the 7 CE were collinearly assembled in the order CE2-3-4-5-6-7-1 to avoid a strongly hydrophobic N-terminal CE1, and were connected via short linker sequences designed for efficient proteolytic cleavage [91, 92]. The COT-M p55^(gag) [93] DNA (plasmid 222H) expresses the full-length Gag from an RNA/codon optimized gene. The IL-12 DNA (plasmid AG157) produces the rhesus macaque IL-12 cytokine from an optimized expression vector [94, 95]. The vaccine vector CMVkan [81] is comprised of a plasmid backbone optimized for growth in bacteria, the human cytomegalovirus (CMV) promoter without introns, the optimized p24CE or gag genes, the bovine growth hormone (BGH) polyadenylation site, and the kanamycin resistance gene. Endotoxin-free DNAs (Qiagen, Valencia, Calif.) were prepared according to the manufacturer's protocol.

Example 3—Additional Conserve Element Polypeptides

Alternative conserved elements were designed (FIG. 13). Briefly, CE1 was extended to provide a CE8. CE2 was extended to provide CE9. In this construct CE7 was removed. Accordingly, there are six conserve elements in the CE polypeptide. There were two variants of the conserve element polypeptide where one amino acid is changed in CE8 and CE9. Two versions were designed having different arrangements of the CEs. In the version termed “p24CEc” (p24CE1c and p24Ce2c), the order is CE8-9-2-3-4-6. In the version terms “p24CEd” (p24CE1d and p24CE2d), the order is CE9-3-4-5-6-8.

Example 4. Illustrative Data from 3 Different Vaccination Prime-Boost Schedules

We tested three different vaccination strategies in which the order of conserved element vaccines and full-length gag vaccine was varied. The three protocols are shown in FIG. 20:

-   1. p24CE prime followed by p55gag boost -   2. p55gag prime followed by p24CE boost -   3. combination of p24CE and p55gag in all vaccinations.

The animals received 1 mg of each DNA. For p24CE, SP-p24CE1 and SPp24CE2 were used. The DNA was administered via the intramuscular route followed by in vivo electroporation. FIG. 21 shows the cellular immune responses before and after the boost. Cellular immune responses were measured with peptides (15-mer overlapping by 11 amino acids) spanning the complete p24gag. This analysis showed responses in all the vaccinated animals.

The animals were also analyzed for CE-specific responses using a peptide pool (mixture of 10-mer peptide overlapping by 9 amino acids and 15-mer overlapping by 11 amino acids) spanning the 7 CE. All the p24CE vaccinated animals showed positive responses. In contrast, Only 5 of the 11 gag DNA vaccinated animals showed responses (data shown). Three of 4 animals with the combination vaccine (p24CE+p55^(gag)) showed positive responses. After boosting by p55gag DNA, the p24CE primed animals significantly increased CE-specific responses. p24CE DNA boost increased the responses of the gag vaccinated animals (no significant increase). A third vaccination of the animals vaccinated by the combination (p24CE+p55^(gag)) did not consistently further increase responses.

FIG. 22 shows the analysis of the responses to individual CE. The responses to each CE were mapped in all the animals using CE-specific peptides (mixture of 10-mer peptide overlapping by 9 amino acids and 15-mer peptides overlapping by 11 amino acids) for each CE. The number of CE that showed positive responses per animal are shown. The p24CE primed animals had 100% response rates with a range of 1-3 CE/animal and median of 3 CE per animal. The gag DNA primed animals have a response rate of 45% with a range of 0-2 CE/animal. The animals that received the combination vaccine are more similar to the CE primed animals.

FIG. 23 shows that different vaccine strategies induced similar levels of p27gag antibody responses. Binding antibody titers were measured in the plasma of macaques by ELISA.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

All publications, accession numbers, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

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Table of Illustrative Conserved Element Sequences p24 Gag conserved elements for p24CE1 vaccine (“also referred to as “Core1”): SEQ ID NO: 1 conserved element 1 (CE1) ISPRTLNAWVKV SEQ ID NO: 2 conserved element 2 (CE2) VIPMFSALSEGATPQDLN SEQ ID NO: 3 conserved element 3 (CE3) VGGHQAAMQMLKDTINEEAAEWDR SEQ ID NO: 4 conserved element 4 (CE4) PRGSDIAGTTSTLQEQIGW SEQ ID NO: 5 conserved element 5 (CE5) KRWIILGLNKIVRMYSPTSI SEQ ID NO: 6 conserved element 6 (CE6) YVDRFYKTLRAEQA SEQ ID NO: 7 conserved element 7 (CE7) LEEMMTACQGVGGPGHK p24 Gag conserved elements for p24CE2 vaccine (“also referred to as “Core2”): SEQ ID NO: 8 conserved element 1 (CE1) LSPRTLNAWVKV SEQ ID NO: 9 conserved element 2 (CE2) VIPMFTALSEGATPQDLN SEQ ID NO: 10 conserved element 3 (CE3) VGGHQAAMQMLKETINEEAAEWDR SEQ ID NO: 11 conserved element 4 (CE4) PRGSDIAGTTSTLQEQIAW SEQ ID NO: 12 conserved element 5 (CE5) KRWIILGLNKIVRMYSPVSI SEQ ID NO: 13 conserved element 6 (CE6) YVDRFFKTLRAEQA SEQ ID NO: 14 conserved element 7 (CE7) LEEMMTACQGVGGPSHK SEQ ID NO: 15 p24 Gag conserved elements for p24CE1 vaccine (“also referred to as “Core1”): VIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAG TTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEEM MTACQGVGGPGHKAAISPRTLNAWVKV SEQ ID NO: 16 p24 Gag conserved elements for p24CE2 vaccine (“also referred to as “Core2”): VIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAG TTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEM MTACQGVGGPSHKAALSPRTLNAWVKV SEQ ID NO: 17 Nucleic acid construct encoding Corel plus Core 2 (p24CE1 + p24CE2) (306H)(genes underlined) CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTAT GCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG TCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATA GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCC CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAG CTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCT CCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGcgcgcgtcgacaagaaATGTGGC TCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGGTCATCCCGAT GTTCTCGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCGTCGG AGGTCACCAGGCAGCGATGCAGATGCTGAAGGACACGATCAACGAGGAGGCGG CCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCA CCTCGACGCTCCAGGAGCAGATCGGGTGGGCCGCAGCTAAGCGCTGGATCATCC TCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGACGTCGATCGCTGCTAAGT ACGTTGACCGGTTCTACAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTGGAGG AGATGATGACCGCGTGCCAGGGGGTCGGTGGACCAGGGCACAAGGCCGCGATCT CGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAAgaattcgctagcggcgcgccagatct gatatcggatctGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGC CTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGA AATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGG CAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGC GGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCC AGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGT TCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTC AATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCA AACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAA GTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATC ATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTG CGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAA CCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAG CATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAA AGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCT GCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT CGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGT GGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAAC CACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGA ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGA TCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGG TCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCA TCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGAC CAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAA GATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCG CCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATT CTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGG ATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCA CCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTC GTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAG TGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATG CATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCA CTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATA CGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCA GGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAA TACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCA GGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTT CAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCT GATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGT TGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAAC ACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATAT TTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGATCATCCA GACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGA AAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTAT AAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTT CAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGT ATGGCTGATTATGATCgtcgaggatccggcgTTATCAGACCTTCACCCAGGCGTTGAGGG TGCGAGGCGAGAGGGCCGCCTTGTGCGACGGTCCTCCGACTCCCTGGCAGGCTGT CATCATCTCCTCGAGACCCGCGGCCTGCTCTGCCCTCAGCGTCTTGAAGAAGCGG TCTACGTATTTGGCCGCGATGCTGACTGGGCTGTACATCCTGACGATCTTGTTGA GGCCCAGGATGATCCAGCGCTTGGCTGCAGCCCAGGCGATCTGCTCCTGGAGGG TGCTGGTCGTGCCTGCGATGTCGCTACCCCTTGGCTCAGCTGCTGCCCTGTCCCAC TCGGCTGCCTCCTCGTTGATGGTCTCCTTGAGCATCTGCATTGCCGCCTGGTGTCC ACCGACCGCGGCGTTGAGGTCCTGCGGTGTCGCACCCTCACTGAGTGCGGTGAAC ATGGGGATGACCGAGATCGAGCACGCCACGGTCCCGAGTAGCAGGAGCGACTGC AGCCACATttcttccgtttaaacgtcgacagatccaaacGCTCCTCCGACGTCCCCAGGCAGAATGG CGGTTCCCTAAACGAGCATTGCTTATATAGACCTCCCATTAGGCACGCCTACCGC CCATTTACGTCAATGGAACGCCCATTTGCGTCATTGCCCCTCCCCATTGACGTCA ATGGGGATGTACTTGGCAGCCATCGCGGGCCATTTACCGCCATTGACGTCAATGG GAGTACTGCCAATGTACCCTGGCGTACTTCCAATAGTAATGTACTTGCCAAGTTA CTATTAATAGATATTGATGTACTGCCAAGTGGGCCATTTACCGTCATTGACGTCA ATAGGGGGCGTGAGAACGGATATGAATGGGCAATGAGCCATCCCATTGACGTCA ATGGTGGGTGGTCCTATTGACGTCAATGGGCATTGAGCCAGGCGGGCCATTTACC GTAATTGACGTCAATGGGGGAGGCGCCATATACGTCAATAGGACCGCCCATATG ACGTCAATAGGTAAGACCATGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACG GTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGC GGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTG TCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT ATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTG GCTATTGGCATTATGCC SEQ ID NO: 18 p24CE1 encoded by SEQ ID NO: 17 (includes a GM-CSF signal peptide) MWLQSLLLLGTVACSISVIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEA AEWDRAAAEPRGSDIAGTTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVD RFYKTLRAEQAAGLEEMMTACQGVGGPGHKAAISPRTLNAWVKV SEQ ID NO: 19 p24CE2 encoded by SEQ ID NO: 17 (includes a GM-CSF signal peptide) MWLQSLLLLGTVACSISVIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEA AEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVD RFFKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKV SEQ ID NO: 20 LAMP-p24CE2 (202H)(gene underlined) CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTAT GCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG TCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATA GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCC CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAG CTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCT CCATAGAAGACACCGGGACCGATCCAGCCTCCGcgggcgcgcgtcgactagcATGGCGCCC CGCAGCGCCCGGCGACCCCTGCTGCTGCTACTGCTGTTGCTGCTGCTCGGCCTCA TGCATTGTGCGTCAGCAGCAATGTTTATGGTGAAAAATGGCAACGGGACCGCGT GCATAATGGCCAACTTCTCTGCTGCCTTCTCAGTGAACTACGACACCAAGAGTGG CCCTAAGAACATGACCCTTGACCTGCCATCAGATGCCACAGTGGTGCTCAACCGC AGCTCCTGTGGAAAAGAGAACACTTCTGACCCCAGTCTCGTGATTGCTTTTGGAA GAGGACATACACTCACTCTCAATTTCACGAGAAATGCAACACGTTACAGCGTTCA GCTCATGAGTTTTGTTTATAACTTGTCAGACACACACCTTTTCCCCAATGCGAGCT CCAAAGAAATCAAGACTGTGGAATCTATAACTGACATCAGGGCAGATATAGATA AAAAATACAGATGTGTTAGTGGCACCCAGGTCCACATGAACAACGTGACCGTAA CGCTCCATGATGCCACCATCCAGGCGTACCTTTCCAACAGCAGCTTCAGCAGGGG AGAGACACGCTGTGAACAAGACAGGCCTTCCCCAACCACAGCGCCCCCTGCGCC ACCCAGCCCCTCGCCCTCACCCGTGCCCAAGAGCCCCTCTGTGGACAAGTACAAC GTGAGCGGCACCAACGGGACCTGCCTGCTGGCCAGCATGGGGCTGCAGCTGAAC CTCACCTATGAGAGGAAGGACAACACGACGGTGACAAGGCTTCTCAACATCAAC CCCAACAAGACCTCGGCCAGCGGGAGCTGCGGCGCCCACCTGGTGACTCTGGAG CTGCACAGCGAGGGCACCACCGTCCTGCTCTTCCAGTTCGGGATGAATGCAAGTT CTAGCCGGTTTTTCCTACAAGGAATCCAGTTGAATACAATTCTTCCTGACGCCAG AGACCCTGCCTTTAAAGCTGCCAACGGCTCCCTGCGAGCGCTGCAGGCCACAGTC GGCAATTCCTACAAGTGCAACGCGGAGGAGCACGTCCGTGTCACGAAGGCGTTT TCAGTCAATATATTCAAAGTGTGGGTCCAGGCTTTCAAGGTGGAAGGTGGCCAGT TTGGCTCTGTGGAGGAGTGTCTGCTGGACGAGAACAGCCTCGAGGATATCGTCAT CCCGATGTTCACGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGC CGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGAGACGATCAACGAGG AGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGG GCACCACCTCGACGCTCCAGGAGCAGATCGCGTGGGCCGCAGCTAAGCGCTGGA TCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGGTCTCGATCGCTGC TAAGTACGTTGACCGGTTCTTCAAGACCCTGAGGGCCGAGCAGGCGGCCGGACT GGAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCATCGCACAAGGCCGC GCTCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCGGATCCGAATTCACGCT GATCCCCATCGCTGTGGGTGGTGCCCTGGCGGGGCTGGTCCTCATCGTCCTCATC GCCTACCTCGTCGGCAGGAAGAGGAGTCACGCAGGCTACCAGACTATCTAGggtac ctctagGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTG CCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGG AAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATG CGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGC CAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGG TTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTT CAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACC AAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTA AGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAAT CATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCT GCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATC AGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGA ACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA AAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTC TCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACT ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCAC TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA GTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTG CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAA ACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTG GAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTC ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATG AGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGGCGCTG AGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCA TCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTG GACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGG GAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAG CCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCA ATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATC AGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAAC TCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGA CTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATC AAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTT ATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAA TCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAA ATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGC GCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTC TAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCA TCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGC CAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATG TTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCA CCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCA TGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCAT AACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATA TATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCC CCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG AAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA AATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAA ACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGC CGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGG TGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATT GG SEQ ID NO: 21 LAMP-p24CE2 fusion (p24CE2 underlined) encoded by SEQ ID NO: 20 MAPRSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFSAAFSVNYDT KSGPKNMTLDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSV QLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTL HDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKYNVSGTNG TCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTVL LFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEH VRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSLEDIVIPMFTALSEGATPQ DLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAA KRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEMMTACQGVGGPSHK AALSPRTLNAWVKVGSEFTLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI. SEQ ID NO: 22 LAMP-p24CE1 (191H)(gene underlined) CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTAT GCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG TCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATA GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCC CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAG CTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCT CCATAGAAGACACCGGGACCGATCCAGCCTCCGcgggcgcgcgtcgactagcATGGCGCCC CGCAGCGCCCGGCGACCCCTGCTGCTGCTACTGCTGTTGCTGCTGCTCGGCCTCA TGCATTGTGCGTCAGCAGCAATGTTTATGGTGAAAAATGGCAACGGGACCGCGT GCATAATGGCCAACTTCTCTGCTGCCTTCTCAGTGAACTACGACACCAAGAGTGG CCCTAAGAACATGACCCTTGACCTGCCATCAGATGCCACAGTGGTGCTCAACCGC AGCTCCTGTGGAAAAGAGAACACTTCTGACCCCAGTCTCGTGATTGCTTTTGGAA GAGGACATACACTCACTCTCAATTTCACGAGAAATGCAACACGTTACAGCGTTCA GCTCATGAGTTTTGTTTATAACTTGTCAGACACACACCTTTTCCCCAATGCGAGCT CCAAAGAAATCAAGACTGTGGAATCTATAACTGACATCAGGGCAGATATAGATA AAAAATACAGATGTGTTAGTGGCACCCAGGTCCACATGAACAACGTGACCGTAA CGCTCCATGATGCCACCATCCAGGCGTACCTTTCCAACAGCAGCTTCAGCAGGGG AGAGACACGCTGTGAACAAGACAGGCCTTCCCCAACCACAGCGCCCCCTGCGCC ACCCAGCCCCTCGCCCTCACCCGTGCCCAAGAGCCCCTCTGTGGACAAGTACAAC GTGAGCGGCACCAACGGGACCTGCCTGCTGGCCAGCATGGGGCTGCAGCTGAAC CTCACCTATGAGAGGAAGGACAACACGACGGTGACAAGGCTTCTCAACATCAAC CCCAACAAGACCTCGGCCAGCGGGAGCTGCGGCGCCCACCTGGTGACTCTGGAG CTGCACAGCGAGGGCACCACCGTCCTGCTCTTCCAGTTCGGGATGAATGCAAGTT CTAGCCGGTTTTTCCTACAAGGAATCCAGTTGAATACAATTCTTCCTGACGCCAG AGACCCTGCCTTTAAAGCTGCCAACGGCTCCCTGCGAGCGCTGCAGGCCACAGTC GGCAATTCCTACAAGTGCAACGCGGAGGAGCACGTCCGTGTCACGAAGGCGTTT TCAGTCAATATATTCAAAGTGTGGGTCCAGGCTTTCAAGGTGGAAGGTGGCCAGT TTGGCTCTGTGGAGGAGTGTCTGCTGGACGAGAACAGCCTCGAGGATATCGTCAT CCCGATGTTCTCGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGC CGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGACACGATCAACGAGGA GGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGG CACCACCTCGACGCTCCAGGAGCAGATCGGGTGGGCCGCAGCTAAGCGCTGGAT CATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGACGTCGATCGCTGCT AAGTACGTTGACCGGTTCTACAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTG GAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCAGGGCACAAGGCCGC GATCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCGGATCCGAATTCACGCT GATCCCCATCGCTGTGGGTGGTGCCCTGGCGGGGCTGGTCCTCATCGTCCTCATC GCCTACCTCGTCGGCAGGAAGAGGAGTCACGCAGGCTACCAGACTATCTAGggtac ctctagGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTG CCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGG AAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATG CGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGC CAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGG TTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTT CAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACC AAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTA AGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAAT CATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCT GCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATC AGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGA ACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA AAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTC TCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACT ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCAC TGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA GTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTG CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAA ACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTG GAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTC ACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATG AGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC GATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGGCGCTG AGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCA TCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTG GACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGG GAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAG CCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCA ATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATC AGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAAC TCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGA CTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATC AAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTT ATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAA TCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAA ATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGC GCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTC TAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCA TCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGC CAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATG TTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCA CCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCA TGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCTCAT AACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATA TATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCC CCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACA TATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG AAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA AATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAA ACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGC CGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGG CTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGG TGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATT GG SEQ ID NO: 23 LAMP-p24CE1 (p24CE1 underlined) MAPRSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFSAAFSVNYDT KSGPKNMTLDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSV QLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTL HDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKYNVSGTNG TCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTVL LFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEH VRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSLEDIVIPMFSALSEGATPQ DLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIGWAAA KRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEEMMTACQGVGGPGHK AAISPRTLNAWVKVGSEFTLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI. SEQ ID NO: 24 SP-p24CE2 (235H)(gene underlined) CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTAT GCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG TCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATA GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCC CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAG CTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCT CCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGcgcgcgtcgacaagaaATGTGGC TCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGGTCATCCCGAT GTTCACGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCGTCGG AGGTCACCAGGCAGCGATGCAGATGCTGAAGGAGACGATCAACGAGGAGGCGG CCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCA CCTCGACGCTCCAGGAGCAGATCGCGTGGGCCGCAGCTAAGCGCTGGATCATCC TCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGGTCTCGATCGCTGCTAAGTA CGTTGACCGGTTCTTCAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTGGAGGA GATGATGACCGCGTGCCAGGGGGTCGGTGGACCATCGCACAAGGCCGCGCTCTC GCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAAgaattcgcggatatcggttaacggatcc AGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCC TTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAA ATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGC AGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCG GTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCA GAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTT CTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCA ATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAA ACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAG TGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCAT AGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCG GCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGG GGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCA TCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAG ATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGC CGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAT AGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGT AACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG TGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGA AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAA AGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAAC GAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCT AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATC TGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTC TGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATC CAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCA GTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGA TGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCC GTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCT GATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGAT TATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACC GAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGT CCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTG AGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCA TTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTC GCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGC GATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGA ACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATAC CTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGA GTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTT AGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAG AAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGAT TGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGG AATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACC CCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTT TATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCCC CCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTG AATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAG TGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAG GCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTC TGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGG AGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGG CTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTG AAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATTGG SEQ ID NO: 25 SP-p24CE2 (p24CE2 underlined) encoded by SEQ ID NO: 24 MWLQSLLLLGTVACSISVIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEA AEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVD RFFKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKV SEQ ID NO: 26 MCP3-p24CE1 (230H)(gene underlined) CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTAT GCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG TCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATA GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCC CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAG CTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCT CCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGcgcgcgtcgacaagaaATGTGG AAGCCGATGCCCTCGCCAAGCAACATGAAGGCGTCCGCCGCGCTCCTGTGCCTGC TCCTCACGGCCGCGGCTTTCAGCCCCCAGGGGCTCGCGCAGCCGGTCGGGATCAA CACGAGCACGACCTGCTGCTACCGGTTCATCAACAAGAAGATCCCGAAGCAGCG TCTGGAGAGCTACCGCCGGACCACGTCGAGCCACTGCCCGCGGGAGGCGGTCAT CTTCAAGACGAAGCTGGACAAGGAGATCTGCGCCGACCCGACGCAGAAGTGGGT TCAGGACTTCATGAAGCACCTGGACAAGAAGACGCAGACGCCGAAGCTGGTCAT CCCGATGTTCTCGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGC CGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGACACGATCAACGAGGA GGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGG CACCACCTCGACGCTCCAGGAGCAGATCGGGTGGGCCGCAGCTAAGCGCTGGAT CATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGACGTCGATCGCTGCT AAGTACGTTGACCGGTTCTACAAGACCCTGAGGGCCGAGCAGGCGGCCGGACTG GAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCAGGGCACAAGGCCGC GATCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAAgaattcgcggatatcggtt aacggatccaGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCC GTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATG AGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGT GGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGG ATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTG GGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCC TGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGC CTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCC ACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTA TTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGA AATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCG GCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGA ATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGA CGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAG CTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGC CACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT GAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCT CTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAAC AAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATAT ATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTC AGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGC TGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCC CATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGG TGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCG GGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAA GCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACC AATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATAT CAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTAT CAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCT TATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAA ATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGA AATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGG CGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTT CTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATC ATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAG CCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCAT GTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGC ACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCC ATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCA TAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGAT ATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTC CCCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATAC ATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCC GAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAA AAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAA AACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATG CCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGG GCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCG GTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTAT TGG SEQ ID NO: 27 MCP3-p24CE1 (p24CE1 underlined) encoded by SEQ ID NO: 26 MWKPMPSPSNMKASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQR LESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL VIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAG TTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEEM MTACQGVGGPGHKAAISPRTLNAWVKV.. SEQ ID NO: 28 MCP3-p24CE2 (231H)(gene underlined) CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCA TGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATC AATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTT ACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGATGGTAAATGGCCCGCCTGGCATTAT GCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG TCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATA GCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCC CATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAG CTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCT CCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGcgcgcgtcgacaagaaATGTGG AAGCCGATGCCCTCGCCAAGCAACATGAAGGCGTCCGCCGCGCTCCTGTGCCTGC TCCTCACGGCCGCGGCTTTCAGCCCCCAGGGGCTCGCGCAGCCGGTCGGGATCAA CACGAGCACGACCTGCTGCTACCGGTTCATCAACAAGAAGATCCCGAAGCAGCG TCTGGAGAGCTACCGCCGGACCACGTCGAGCCACTGCCCGCGGGAGGCGGTCAT CTTCAAGACGAAGCTGGACAAGGAGATCTGCGCCGACCCGACGCAGAAGTGGGT TCAGGACTTCATGAAGCACCTGGACAAGAAGACGCAGACGCCGAAGCTGGTCAT CCCGATGTTCACGGCGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGC CGTCGGAGGTCACCAGGCAGCGATGCAGATGCTGAAGGAGACGATCAACGAGG AGGCGGCCGAGTGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGG GCACCACCTCGACGCTCCAGGAGCAGATCGCGTGGGCCGCAGCTAAGCGCTGGA TCATCCTCGGGCTGAACAAGATCGTCCGGATGTACAGCCCGGTCTCGATCGCTGC TAAGTACGTTGACCGGTTCTTCAAGACCCTGAGGGCCGAGCAGGCGGCCGGACT GGAGGAGATGATGACCGCGTGCCAGGGGGTCGGTGGACCATCGCACAAGGCCGC GCTCTCGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAAgaattcgcggatatcggtt aacggatccaGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCC GTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATG AGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGT GGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGG ATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTG GGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCC TGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGC CTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCC ACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTA TTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGA AATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCG GCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGA ATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGA CGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAG CTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGC CACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTT GAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCT CTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAAC AAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG AAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATAT ATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTC AGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGC TGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCC CATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGG TGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCG GGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAA GCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACC AATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATAT CAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAA CTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTAT CAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCT TATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAA ATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGA AATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGG CGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTT CTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATC ATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAG CCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCAT GTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGC ACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCC ATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCA TAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGAT ATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTC CCCCCCCCCCCATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATAC ATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCC GAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAA AAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAA AACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATG CCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGG GCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCG GTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTAT TGG SEQ ID NO: 29 MCP3-p24CE2 (p24CE2 is underlined) encoded by SEQ ID NO: 28 MWKPMPSPSNMKASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQR LESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL VIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAG TTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEM MTACQGVGGPSHKAALSPRTLNAWVKV. SEQ ID NO: 30 SP-p24CE1c-alternative conserved element nucleic acid construct ATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGCAGG GGCAGATGGTCCACCAGGCGATCTCGCCGCGCACGCTGAACGCGTGGGTGAAGG TCCTGGCGAAGGAGGAGAAGGCGTTCAGCCCGGAGGTCATCCCGATGTTCTCGG CGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCAAGGTCGGAGGTC ACCAGGCAGCGATGCAGATGCTGAAGGAGACGATCAACGAGGAGGCGGCCGAG TGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCG ACGCTCCAGGAGCAGATCGGGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGG CTGAACAAGATCGTCCGGATGTACAGCCCGACGTCGATCGCTGCTAAATACGTTG ACCGGTTCTACAAGACCCTGAGGGCCGAGCAGGCGGATTACAAGGACGATGACG ACAAGCTGTGATAA SEQ ID NO: 31 SP-p24CElc (p24CElc underlined) encoded by SEQ ID NO: 30. Includes GM-CSF signal peptide, CE1 and CE2 replaced by CE8 and CE9, respectively (relative to p24 CE “Core1”); lacks CE7; arranged in the configuration of conserved elements: CE 8-9-3-4-5-6 MWLQSLLLLGTVACSISQGQMVHQAISPRTLNAWVKVLAKEEKAFSPEVIPMFSALS EGATPQDLNAAKVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQE QIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQADYKDDDDKL SEQ ID NO: 32 conserved element 8 (CE8) QGQMVHQAISPRTLNAWVKV SEQ ID NO: 33 conserved element 9 (CE9) EEKAFSPEVIPMFSALSEGATPQDLN SEQ ID NO: 34 SP-p24CE2c-alternative ATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGCAGG GGCAGATGGTCCACCAGGCGCTGTCGCCGCGCACGCTGAACGCGTGGGTGAAGG TCCTGGCGAAGGAGGAGAAGGGGTTCAACCCGGAGGTCATCCCGATGTTCACGG CGCTCAGCGAGGGAGCGACGCCGCAGGACCTGAACGCGGCCAAGGTCGGAGGTC ACCAGGCAGCGATGCAGATGCTGAAGGACACGATCAACGAGGAGGCGGCCGAG TGGGACCGGGCGGCAGCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCG ACGCTCCAGGAGCAGATCGCGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGG CTGAACAAGATCGTCCGGATGTACAGCCCGGTCTCGATCGCTGCTAAATACGTTG ACCGGTTCTTCAAGACCCTGAGGGCCGAGCAGGCGTGATAA SEQ ID NO: 35 SP-p24CE2c (p24CE2c underlined) MWLQSLLLLGTVACSISQGQMVHQALSPRTLNAWVKVLAKEEKGFNPEVIPMFTAL SEGATPQDLNAAKVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQ EQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQA SEQ ID NO: 36 SP-p24CE2d alternative nucleic acid conserved element nucleic acid construct; in order CE9-3-4-5-6-8 ATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGGAGG AGAAGGGGTTCAACCCGGAGGTCATCCCGATGTTCACGGCGCTCAGCGAGGGAG CGACGCCGCAGGACCTGAACGCGGCCAAGGTCGGAGGTCACCAGGCAGCGATGC AGATGCTGAAGGACACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCA GCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAG ATCGCGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTC CGGATGTACAGCCCGGTCTCGATCGCTGCTAAATACGTTGACCGGTTCTTCAAGA CCCTGAGGGCCGAGCAGGCGGCGCTGCAGGGGCAGATGGTCCACCAGGCGCTGT CGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAA SEQ ID NO: 37 p24CE2d (protein underlined) encoded by SEQ ID NO: 36 MWLQSLLLLGTVACSISEEKGFNPEVIPMFTALSEGATPQDLNAAKVGGHQAAMQM LKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSP VSIAAKYVDRFFKTLRAEQAALQGQMVHQALSPRTLNAWVKV SEQ ID NO: 38 SP-24CE1d-conserved element nucleic acid construct; order in CE9-3-4-5-6-8 ATGTGGCTCCAGAGCCTGCTACTCCTGGGGACGGTGGCCTGCAGCATCTCGGAGG AGAAGGCGTTCAGCCCGGAGGTCATCCCGATGTTCTCGGCGCTCAGCGAGGGAG CGACGCCGCAGGACCTGAACGCGGCCAAGGTCGGAGGTCACCAGGCAGCGATGC AGATGCTGAAGGAGACGATCAACGAGGAGGCGGCCGAGTGGGACCGGGCGGCA GCCGAGCCACGCGGTTCCGACATCGCGGGCACCACCTCGACGCTCCAGGAGCAG ATCGGGTGGGCCGCAGCTAAGCGCTGGATCATCCTCGGGCTGAACAAGATCGTC CGGATGTACAGCCCGACGTCGATCGCTGCTAAATACGTTGACCGGTTCTACAAGA CCCTGAGGGCCGAGCAGGCGGCGCTGCAGGGGCAGATGGTCCACCAGGCGATCT CGCCGCGCACGCTGAACGCGTGGGTGAAGGTCTGATAA SEQ ID NO: 39 SP-24CE1d encoded by SEQ ID NO: 38 MWLQSLLLLGTVACSISEEKAFSPEVIPMFSALSEGATPQDLNAAKVGGHQAAMQM LKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIGWAAAKRWIILGLNKIVRMYSP TSIAAKYVDRFYKTLRAEQAALQGQMVHQAISPRTLNAWVKV p24CE1d has 6 CE (is identical to p24CE1c except for the CE arrangement within the protein) GM-CSF signal peptide CE1 and C2 replaced by CE8 and CE9 respectively, lacks CE7 and has the CE arranged in the configuration CE9-3-4-5-6-8 SEQ ID NO: 40 conserved element 8 (CE8-variant for CE2 constructs) QGQMVHQALSPRTLNAWVKV SEQ ID NO: 41 conserved element 9 (CE9) EEKGFNPEVIPMFTALSEGATPQDLN

Differences between CE for p24 CE polypeptides and variant p24CE polypeptides is one amino acid per CE except CE9, which differs by 3 amino acids 

What is claimed is:
 1. A method of inducing an immune response to an HIV protein, the method comprising administering: a first nucleic acid encoding a first conserved element polypeptide, wherein the conserved elements are from the HIV protein and the polypeptide comprises at least six conserved elements, each less than 50 amino acids in length, where the conserved elements are joined by linkers; a second nucleic acid encoding a second conserved element polypeptide that comprises a variant of each of the at least six conserved elements contained in the first conserved element polypeptide, wherein each variant in the second conserved element polypeptide differs from the corresponding conserved element in the first conserved element polypeptide by at least 1, 2, or 3 amino acids; and after administration of the first and the second nucleic acids, administering a nucleic acid encoding the full-length HIV protein, or substantially full-length HIV protein, wherein the nucleic acid encoding the full-length HIV protein or substantially full-length HIV protein is administered at least two weeks after the first and second nucleic acids encoding the conserved element polypeptides; and further, wherein the nucleic acid encoding the full-length HIV protein, or substantially full-length HIV protein, is administered as a plasmid that comprises an expression cassette comprising a nucleic acid sequence encoding the full-length HIV protein, or substantially full-length HIV protein, operably linked to a promoter.
 2. The method of claim 1, wherein the HIV protein is an HIV Gag protein or an HIV Env protein.
 3. The method of claim 2, wherein the variants of the at least six conserved elements each have 80% amino acid sequence identity to its corresponding conserved element in the first conserved element polypeptide.
 4. The method of claim 3, wherein the first conserved element polypeptide comprises at least one, two, three, four five, or six conserved elements that have an amino acid sequence set forth in SEQ ID NOS:1-7, 32, or
 33. 5. The method of claim 2, wherein the first conserved element polypeptide comprises conserved elements that each have a sequence set forth in SEQ ID NOS:1-7; or the first conserved element polypeptide comprises conserved elements that each have a sequence set forth in SEQ ID NOS:3-6, 32, and
 33. 6. The method of claim 2, wherein the second conserved element polypeptide comprises at least one conserved element that has an amino acid sequence set forth in SEQ ID NOS:8-14, 40, or
 41. 7. The method of claim 2, wherein the second conserved element polypeptide comprises at least two, three, four five, or six conserved elements that have an amino acid sequence set forth in SEQ ID NOS:8-14, 40, or
 41. 8. The method of claim 2, wherein the second conserved element polypeptide comprises conserved elements that each have a sequence set forth in SEQ ID NOS:8-14; or the second conserved element polypeptide comprises conserved elements that each have a sequence set forth in SEQ ID NOS:10-13, 40 and
 41. 9. The method of claim 2, wherein the conserved element and variant conserved element each have a sequence set forth in FIG. 1 or FIG.
 13. 10. The method of claim 2 wherein the first conserved element nucleic acid encodes a conserved element polypeptide comprising the sequence of SEQ ID NO:15 and the second conserved element nucleic acid encodes a conserved element polypeptide comprising the sequence of SEQ ID NO:16.
 11. The method of claim 1, wherein the first and second conserved element nucleic acids are administered sequentially.
 12. The method of claim 1, wherein the first and second nucleic acid conserved element nucleic acids are administered concurrently.
 13. The method of claim 1, wherein the conserved element polypeptides encoded by the first and second nucleic acid conserved element nucleic acids are each fused to a GM-CSF signal peptide.
 14. The method of claim 1, wherein the first and second nucleic acid conserved element nucleic acids are contained in the same vector.
 15. The method of claim 1, wherein the first and second nucleic acid conserved element nucleic acids are contained in different vectors.
 16. The method of claim 1, wherein the nucleic acid constructs encoding the conserved element polypeptides and full-length, or substantially full-length, polypeptide are administered intramuscularly by in vivo electroporation.
 17. The method of claim 1, wherein the protein of interest is HIV gag and the first and the second conserved element polypeptides each comprise seven conserved elements.
 18. The method of claim 17, wherein the first conserved element polypeptide comprises the amino acid sequences of SEQ ID NOS:1, 2, 3, 4, 5, 6, and 7; and the second conserved element polypeptide comprises the amino acid sequences of SEQ ID NOS:8, 9, 10, 11, 12, 13, and
 14. 